Incidence of Helicobacter Pylori Infections in a group of patients in Erbil City, Kurdistan Region, Iraq and Its Association with Gastritis and Adenocarcinoma

Background: Adenocarcinoma is one of the most common causes of Gastric cancer related deaths worldwide. Helicobacter pylori is the causative agent of most cases of gastritis, it can cause chronic active gastritis and known as a risk factor for the development of gastric cancer. This study aimed to assess the prevalence of H. pylori among patients with symptoms of dyspepsia and other gastritis related symptoms and its association with adenocarcinoma. Methods: This study was carried out during the period of January 2018 to October 2019 with a total of 227 patients with gastritis related symptoms. The presence of H. pylori was detected by Rapid Urease Test (RUT) and histo-pathological tests using biopsy specimens. Statistical Analysis was done by using Chi-square test. P < 0.05 was considered to be statistically signi�cant. Results: From the total of 227 patients with gastritis related symptoms, 26 cases (13.61%) were diagnosed with adenocarcinoma. Their ages were between 13 and 90 years with mean of 47.81± 18.23. The result showed that low severity prevalence of H. pylori was highest (111 cases) compared to 17 and 63 cases for high and moderate severity, respectively. Comparison between positive low, moderate, and high H. pylori cases for rapid urease test was highly signi�cant (P<0.000). The results showed no association between H. pylori severity across various age groups and gender. Moreover, goodness of �t test for metaplasia, activity, glandular atrophy, and endoscopic �nding across severity status of H. pylori showed highly signi�cant. Four composite categorized groups were initiated based on positive/negative prevalance of H. pylori and adenocarcinoma status. Results revealed statistical signi�cance between combination of H. pylori and adenocarcinoma with in�ammation, lymphoid aggregate, metaplasia, activity of neutrophils, glandular atrophy, rapid urease test, and endoscopic �ndings. Conclusion: Histopathology tests are reliable diagnostic tools for the detection of H. pylori. Data showed that


Background
The discovery of Helicobacter pylori (H. pylori) in 1982 was the starting point of a revolution concerning the concepts and management of gastroduodenal diseases. 1,2 H. pylori represents one of the most common and medically important infections worldwide. 3 Chronic infection due to this pathogen is now believed to account for the majority of cases of chronic gastritis. 4 In addition, H. pylori is the major cause of gastric carcinogenesis and other gastric diseases, such as duodenal and gastric non-ulcer dyspepsia, gastroduodenal ulcers, and gastric mucosa-associated lymphoid tissue lymphoma. 1,5 In recent years, a variety of extra digestive disorders, including cardiovascular diseases, liver diseases and autoimmune disorders have also been associated with infections caused by H. pylori. 6 Gastric cancer is the third most common cause of death caused and related to cancer worldwide. H. pylori infection is the most known risk factor for the development of gastric cancer. The pathogen can cause chronic active gastritis and atrophic gastritis by producing persistent acute-on-chronic in ammation. 7 The high association of H. pylori infections with precancerous gastric lesions led to the pathogen being recognized as a class I carcinogen by the WHO in 1994. 8 It has also been reported that in a meta-analysis infections, caused by H. pylori, results in a 2-to 3-fold increase in the risk of gastric cancer. 9 It is estimated that approximately 75% of the gastric cancer burden in the world is attributed to H. pylori-induced in ammation. 10 This study aimed to assess the prevalence of H. pylori among patients with symptoms of dyspepsia and other gastritis related symptoms. It also aimed to investigate the association between H. pylori with adenocarcinoma.

Methods
This study was conducted in the Histopathology laboratory of PAR Private Hospital, Erbil, Iraq, within the period of January 2018 to October 2019. A total of 227 Patients were included of any age and gender with symptoms of gastritis. A questionnaire regarding the age, gender and whether they were taking any dyspepsia related medications was included. The urea breath test was used rst to detect H. pylori, in which the principle of the test is the pathogen can produce an enzyme called urease, which breaks urea down into ammonia and carbon dioxide. During the test, a tablet containing urea is swallowed and the amount of exhaled carbon dioxide is measured. This indicates the presence of H. pylori in the stomach (BreathTek® UBT for H. pylori kit was used). Three biopsy specimens from the antrum and the corpus were taken and sent for histopathologic study. In the histopathological unit, the biopsy specimens of patients were xed in 10% buffered formalin for at least 12 hrs, and then embedded in para n wax.
Hematoxylin and Eosin (H and E) staining was performed on tissue sections of each case. Three sections for each specimen were de-para nized and hydrated in descending grades of alcohol, cut in sequential 4µm sections. The sections were then stained with H and E stain to determine the presence of H. pylori and gastritis. The H. pylori were clearly detected as curved bacilli on the surface of the gastric epithelial cells. The slides were evaluated by histopathologist and assigned to each morphological variable. The histopathology was used to con rm gastritis and neoplastic conditions found during endoscopy.
The positive and negative results of H. pylori infection were categorized together with poor and moderate grades adenocarcinoma status according to the age group, gender and different diagnosis variables {positive H. pylori/ moderate grade differentiated adenocarcinoma (positive / moderate), positive H. pylori/ poor grade differentiated adenocarcinoma (positive/ poor), negative H. pylori/ moderate grade differentiated adenocarcinoma (negative/ moderate) and negative H. pylori / poor grade differentiated adenocarcinoma (negative / poor)}.

Statistical Analysis
All variables of the patients were entered into Excel worksheet and data analysis was performed using Chi-square independence test, via contingency tables (Statistical Package for the Social Sciences software, SPSS Inc., Chicago, IL, USA). As Chi-square test for 2 × 3, 3 × 3 or 3 × 4 contingency tables did not support calculation when zero exists as a cell value. Therefore, a goodness of t (differences between observed and expected cases) was performed using GraphPad Software. 11 The P value less than 0.05 was considered to be statistically signi cant.

Results
The minimum age of the 227 patients was 13 years, maximum age was 90 years and mean age was 47.81 ± 18.23. Furthermore, the minimum, maximum and mean age of the 191 infected patients with H. pylori based on the categorized severity of the H. pylori into high, moderate and low is presented in Table 1. The low state prevalence of H. pylori was highest (111 cases) compared to 17 and 63 cases for high and moderate severity of H. pylori, respectively. This result showed that cases with no detectable infection were 36 (15.86%) out of the 227 patients. Results revealed that all patients had positive rapid urease test and no association between H. pylori severity across various age groups (P = 0.245), and gender (P < 0.961). It was noticed that high prevalence of H. pylori was most likely to occur in patients of 35-60 years old (43.98%) compared to younger or older ages (30.89, 25.13%), respectively ( Table 2). The prevalence of H. pylori infection was 50.26% in males 49.74% in females showing similar distribution of H. pylori among the gender. The result showed a statistical signi cant association between H. pylori severity across various In ammation (P < 0.001), and Lymphoid aggregate (P < 0.001). Chi square for diagnosis variables of metaplasia, activity which represented by presence or absence of neutrophils, glandular atrophy, rapid urease test, endoscopic nding, and clinical manifestation were highly signi cant (P < 0.001) across low, moderate, and high H. pylori severity (Fig. 1).
The moderate In ammation was recorded the highest (71.2%) compared to mild (25.66%) and severe (3.14%). The nding of the positive lymphoid aggregate showed 82.72% compared to 17.28% of the negative tests (Fig. 2).
On the other hand, positive metaplasia was found in 11.52%; neutrophils in 17.8% and positive tests for glandular atrophy in 9.95% of cases.
The distribution of the endoscopic ndings showed 64.92% nodular, whereas 35.08% were erosive.

Discussion
The prevalence of H. pylori differs both between and within countries, with high rates of infection being associated with low socioeconomic status and high densities of living. 12 When endoscopy is clinically indicated, the test of rst choice is the urease test on an antral-biopsy specimen. 13 In the current study, the minimum age of the 227 patients was 13 years and the maximum age was 90 years, with mean age of 47.81 ± 18.23. High prevalence of H. pylori was most likely to occur in patients of 35-60 years old (43.98%) compared to younger or older ages (30.89, 25.13%), respectively. The result of this study shows that the prevalence of H. pylori infection found in male was 50.26% and in females was 49.74%, this is similar to the distribution of H. pylori among the gender in patients studied in Nepal. Where among 2820 eligible patients, males were 54.2% and females were 45.8% with the mean age of 46.3 years ± 17.6 ranging from 8 to 94 years. 14 On the other hand, in a large French cross-sectional study, a signi cantly lower prevalence of H. pylori infection was observed in females as compared with males. 15 Several studies were conducted in Kurdistan dealing with prevalence of H. pylori and gastritis. Research result indicated that the prevalence of H. pylori in total of 310 samples (170 male and 140 female) were Similarly, it can be seen that lymphoid aggregate status (positive vs. negative tests) has similar trends across H. pylori severity status (Fig. 5). Both positive and negative lymphoid aggregate showed lowest values (17 and 0, respectively) for high H. pylori and both diagnostic tests increased to 60 and 3 cases for the moderate H. pylori status, then both attained the peak cases of their lines when H. pylori status was low(81 and 30, respectively). Such similar trend plots indicate a high signi cant statistical positive association. Table 3, Figs. 6 and 7 illustrate highly statistically signi cant (P < 0.001) ndings between a combination of positive and negative H. pylori and poor or moderate adenocarcinoma with rapid urease test, and glandular atrophy. Figure 6 shows uctuations in number of cases among categorization (combination) of H. pylori and adenocarcinoma with rapid urease test, which represent an inverse association, as the trend of the positive rapid urease test line has opposite direction and magnitude of the negative rapid urease test line. The positive rapid urease test line increased from 4 to 6 then declined to 0, 0 for (positive / moderate), (positive / poor), (negative / moderate) and (negative / poor), respectively. Whereas, negative rapid urease test line increased from 0, 0 to 5 then to 11 for (positive / moderate), (positive / poor), (negative / moderate) and (negative / poor), respectively.
Similarly, Fig. 7 shows uctuations in number of cases among categorization (combination) of H. pylori and adenocarcinoma with positive and negative glandular atrophy, which represent inverse association, as the trend of the positive glandular atrophy line has opposite direction and magnitude of the negative glandular atrophy line. The negative glandular atrophy line increased from 1 to 2 then to 5and 11 for (positive / moderate), (positive / poor), (negative / moderate) and (negative / poor), respectively. Whereas, positive glandular atrophy line increased from 3, to 4 then declined to 0, 0 for (positive / moderate), (positive / poor), (negative / moderate) and (negative / poor), respectively. 39.4%. The prevalence of H .pylori in the age between 41-50 years were (51.2%), followed by patients with age > 61 years (46.5%). The rate of infection among females were (40.7%), compared with males infection rate of (38.2%). That study concluded that the prevalence of H. pylori in Erbil city was high and the infection occurred at different stages of life. 16 Other report found that the prevalence of H. pylori infection among 311 students (57.8% female and 42.2% male) increased with age and a higher frequency found in students from low income social status. They also observed that H. pylori were highly prevalent among university students in Erbil, being 55.8% of the 311 students. 17 Recently, in a study carried out to investigate the seroprevalence of H. pylori infection among Cihan University students, Erbil. A total of 197 blood samples were collected from the students (53 females and 144 males), and tested for anti-H. pylori antibodies, using a rapid immunochromatography assay. The prevalence of anti-H. pylori antibodies is relatively high among students, with no signi cant differences between male and female in respect to having H. pylori infection. It was concluded that both university and public communities should be aware of infections caused by the pathogen such as gastritis, peptic ulcer, and about its potential association with malignant transformation. 18 Furthermore, investigation of H. pylori infection among 190 patients diagnosed with gastric cancer in Sulaimani city indicated that a history of H. pylori infection was observed in 63.2% of the patients and that prevalence of gastric cancer was higher among females (61.5%), and patients aged over 50 years (73.6%). 19 Interestingly a study carried out on 50 patients suffering from stomach cancer in Erbil city and 25 healthy individuals, as a control group showed that the highest number of chromosomal aberrations (Dicentric chromosome) occurred in the fth age group (65-74). It also shown that most patients are males in the age group (45-64), most of whom smoked and drank alcohol and were infected with H. pylori. 20 The present study also revealed statistically signi cant associations between H. pylori and adenocarcinoma with in ammation, glandular atrophy, and rapid urease test. Furthermore, results revealed highly statistically signi cant (P < 0.001) differences among categorization (combination) of H. pylori and adenocarcinoma with in ammation, lymphoid aggregate, metaplasia, activity of neutrophils, glandular atrophy, endoscopic, and rapid urease test. The result also indicated that 26 cases out of 227 cases (13.61%) were diagnosed to have adenocarcinomia, and that 10 out of 26 adencarcinoma cases (about 38.5%) showed positive rate of H. pylori infection. Similar result of low rate of gastric cancer incidence was noted with high prevalence of H. pylori infection in India, Philippines, or Thailand; on the other hand, the high rate of gastric cancer incidence was observed in Japan and Korea with variable prevalence of H. pylori. [21][22][23] It was suggested that H. pylori infection is not the only factor related to gastric cancer risk. 24,25 Furthermore, Individuals with chronic H. pylori infection have an increased risk of acquiring a adenocarcinoma, 26,27, 28 as the pathogen indicated as a carcinogen associated with gastric adenocarcinoma. 29 Colonization of stomach by H. pylori can result in gastritis, and may have a risk of developing peptic ulcers (Kusters et al., 2006), it is notable that in this study 35.08% of the H. pylori gastritis revealed erosion. The National Institutes of Health consensus conference in the United States declared an association between H. pylori and peptic ulcer disease. 30,31 Data produced from this study showed that low density of H. pylori was highest (111 cases) compared to 17 and 63 cases for high and moderate density infections, respectively. H. pylori caused chronic gastritis in all colonized subjects. This can lead to peptic ulcer disease, atrophic gastritis, gastric adenocarcinoma, and MALT (mucosa-associated lymphoid tissue) lymphoma. 32 Patients with current H. pylori infection have a higher risk of developing gastric cancer compared with patients with past infection or eradication history of H. pylori because the eradication of H. pylori reduces the risk of gastric cancer. It was reported that the successful treatment of H. pylori decreases the risk of developing gastric cancer by approximately 3 fold. 33 Therefore, screening and treatment of H. pylori is an important strategy for preventing gastric cancer in high-risk populations, particularly among societies.
This study has a number of limitations. The generalizability of the ndings is limited as samples were collected only from one city (Erbil), this suggests that the study population might not be representative of the general H. pylory infections in Kurdistan region. The sample size of this study is relatively small for a region with a high burden of the disease as the diagnosed patients are only from one city of the region in Iraq.

Conclusion
The histopathology and RUT tests are reliable invasive diagnosis for H. pylori. It was noticed that H. pylori seen more in middle age group patients living in Erbil ,Kurdistan with mucosal lymphoid follicle formation, moderate active in ammatory changes as well as H. pylori seen in more than one third of patients with adenocarcinoma cases. So screening, follow up and treatment of H. pylori is an important strategy for preventing gastric adenocarcinoma in Erbil, Kurdistan.      Positive association among severity of H. pylori status with lymphoid aggregate status. Inverse association among severity of H. pylori status with rapid urease test. Inverse association among severity of H. pylori status with glandular atrophy test.