Occurrence of Helicobacter Pylori in Specimens of Chronic Gastritis and Gastric Adenocarcinoma Patients: A Retrospective Study at University Teaching Hospital, Kigali, Rwanda

Introduction: Helicobacter pylori (H. pylori) infection is the major cause of gastroduodenal diseases in populations of different ages. We conducted aretrospective studyusing archived tissue samples to determine the prevalence of H. pylori infection among patients diagnosed with gastritis and gastric adenocarcinoma by histopathology cases in one hospital in Rwanda. Materials and methods: Cases of chronic gastritis and gastric adenocarcinoma histologically diagnosed in a tertiary hospital in Rwanda over the period of 2016-2018 were studied for the presence of H. pylori using immunohistochemistry. Diagnosis of positive cases considered immunoreactivity as well as bacterial morphology, including spiral, rod-shaped, angulated and coccoid forms. Results: Three hundred and seven cases were included in this study; chronic gastritis and gastric adenocarcinoma representing 39% and 61%, respectively. The overall frequency of H. pylori infection was 77.5% (80% among chronic gastritis cases versus 76% among gastric adenocarcinoma cases). Prevalence of H. pylori infection in chronic gastritis and adenocarcinoma did not significantly associate with age and sex. Conclusion: The prevalence of H. pylori was high among chronic gastritis and gastric adenocarcinoma cases in Rwanda. Pathologists should investigate the presence of H. pylori in gastric biopsies. Our data shows immunohistochemistry method is feasible and adequate to facilitate detection of H. pylori, which may guide timely treatment.

eventually lead to gastric cancer. 2,13,19,20 In H. pyloriinfected individuals, other factors contributing to chronic atrophic gastritis and cancer include the age at the time of primary infection, as well as the presence of cytotoxin-associated gene A (cagA)-positive H. pylori. 21 The prevalence of H. pylori infection increases with age, being close to 80% among individuals above 70, whereas it is around 50% in children. 20,22,23 Several diagnostic tests are used in the detection of H. pylori infection. These include blood serum test, stool antigen test, rapid urease test, urea breath test, detection of H. pylori in histopathology specimens, and culture. 5,8,11,12,24 Histopathology has been shown to have excellent sensitivity and specificity (95% and 99%, respectively),particularly with the use of special and immunohistochemical stains 25 and it provides additional information about the morphology of the gastric mucosa. 12,[22][23][24] Accordingly, endoscopic biopsiesare used for screening of gastriccarcinoma. 8,16,22,26 The prevalence of gastroduodenal disease in Rwanda is high, and a recent study using modified rapid urea-BACKGROUND H elicobacter pylori (H. pylori) is a gramnegative bacterium that causes a spectrum of gastroduodenal diseases in humans including chronic gastritis and gastric cancer. [1][2][3] Approximately 95% of gastric cancers are adenocarcinomas, which are further histologically categorized into diffuse and intestinal subtypes. 4 The prevalence of H. pylori is approximately 50% of the adult people worldwide. [5][6][7] The prevalence is much higher in populations of low socioeconomic status and hygiene level, compared to the developed countries. [8][9][10][11] Accordingly, the prevalence of H. pylori infection is nearly 30% in the United States of America (USA) adult population, compared to up to 92% in some African regions. 7,[12][13][14] This epidemiological trend may explain the overrepresentation of gastric adenocarcinoma among developing countries (more than 50% of new cases) compared to the developed countries. 4,[15][16][17][18] Chronic atrophic gastritis is the earliest pathologic change due to H. pylori colonization, and it may se testing during endoscopy showed 75% positivity for H. pylori. 27 Using immunohistochemistry method, this study was performed to determine the frequency of occurrence of H. pylori infection in histopathological specimens of patients with chronic gastritis or gastric adenocarcinomain a large teaching hospital of Rwanda.

Study Design and Description
A retrospective descriptive study was conducted in the Anatomical Pathology unit of University Teaching Hospital of Kigali (CHUK). Cases diagnosed as chronic gastritis and gastric adenocarcinoma from 2016 to 2018 were included in this study. Clinical and demographic information and formalin-fixed, paraffin-embedded (FFPE) tissue blocks, and glass slides were retrieved from the archives.
For cases with multiple biopsies from the same patient, the tissue sample with more representative lesion tissue was used. Glass slides were reviewed by 2 independent pathologists to confirm the diagnosis of chronic gastritis or gastric adenocarcinoma. All cases diagnosed as gastric ulcers, gastritis with intestinal metaplasia and/or atrophic gastritis, and chronic gastritis not otherwise specified were included. Sections of tissues with lesion (gastritis or adenocarcinoma) were selected for immunohistochemistry. Patients who met the inclusion criteria, but whose tissue blocks were damaged were excluded.

Helicobacter pylori detection
Sections (4 µm in thickness) were cut and prepared on charged, frosted glass slides. Immunohistochemistry using a rabbit polyclonal anti-H. pylori antibody (DAKO) and the Envision (DAKO) polymer detection system, with diaminobenzidine chromogen and immune-peroxidases according to the manufacturer's specifications was performed. Positive and negative controls were evaluated for each immunostaining assay. Two independent pathologists and one trainee reviewed the immunestained slides using light microscopes. In case of discrepancy, cases were reviewed and discussed, and the consensus diagnosis agreed by all pathologists. Positivity was ascertained taking into account the presence of immunoreactivity and morphology including spiral, rodshaped, angulated, and coccoid forms.

Data management and statistical analysis
Clinical, demographic, and histopathologic diagnosis, including H. pylori status, were compiled into a Microsoft Excel sheet. Each patient was assigned an identification code to maintain patients' confidentiality. The data was imported into and analyzed using the Statistical Product and Service Solutions(IBM SPSS). Fisher's exact test was used to compare proportions. A two-tailed P value <.05 was considered significant.

Patients and Disease Characteristics
Most 197(64.2%) patients with chronic gastritis and gastric adenocarcinoma were older than 50 (range=15 to 92, mean=55, median=57) years (Table 1). All the regions of Rwanda are represented, with a slightly higher proportion of patients residing in Kigali City 72(23.5%). Most 264 (86%) biopsies were endoscopic and almost all (98.4%) were taken from the non-cardial (distal) part of the stomach and 61% of cases were diagnosed with gastric adenocarcinoma, with a predominance of intestinal type gastric adenocarcinoma (Table 1). Figure 1 illustrates the routinely stained sections of gastric adenocarcinoma (Figure 1.A-B) and chronic gastritis (Figure 1.C), as well as the various morphologies of H. pylori as detected using immunohistochemistry (Figure 1.D-F).

Biological behavior of chronic gastritis and gastric adenocarcinoma
The prevalence of H. pylori infection was 80.2% and 75.8% in chronic gastritis and gastric adenocarcinoma, respectively ( Table 2), but there was no significant difference in the number of cases with H. pylori infection when comparing chronic gastritis and gastric adenocarcinoma groups of patients (P=0.371). Moreover, the type of gastric adenocarcinoma showed no correlation with H. pylori infection (P=0.732).
The frequency of presence of H. pylori in the reviewed biopsies did not varies with the types of studied gastroduodenal diseases (chronic gastritis and adenocarcinoma), age and sex ( Table 2).    13,25 In the present study, we analyzed the proportion of H. pylori infection among pathology samples of patients diagnosed with chronic gastritis and gastric adenocarcinomain one of the hospitals in Rwanda.
The high proportion of endoscopic biopsies (86%) in our cohort is in keeping with the fact that endoscopic biopsy is considered a gold standard procedure for the screening and the detection of gastric cancer. 1,20 In both patient groups, the majority of cases were older than 50 years of age, while both sexes were almost equally represented.
The relatively over-representation of Kigali and the Eastern regions among the cohort may be explained by the geographical accessibility to the study site.
The overall prevalence of H. pylori infection among both disease groups was high (77%). Asimilarproportion (75%) of H. pylori infection (using modified rapid urease test) was previously reported in Rwanda, in a study comprising all patients who underwent upper gastrointestinal endoscopy. The proportions, although slightly different, are not significantly different. 27 The proportion of H. pylori infection among various cohorts of individuals in Africa ranges from 55 to 92%. 11,14 In addition, there was no significant difference in the rates of H. pylori infection between chronic gastritis and gastric adenocarcinoma (P= 0.371). These findings are similar to those previously reported in other settings. 14 In the present study, although there was no significant association between age (using a 50-year cut-off) and H. pylori infection, a trend towards a higher proportion of H. pylori infection with increasing age among chronic gastritis patients was observed. In contrast, it tends to de-crease with increasing age among gastric adenocarcinoma patients. These findings are consistent with the previous studies which reported that H. pylori infection is typically universal in all adulthood age groups 20, 28 , because it is up taken during youthfulness and generally persists during lifetime except if correctly managed. 29

CONCLUSION
This study documents a high prevalence of H. pylori infection in pathology specimens at one major hospital in Rwanda. Study findings indicate that all age and both sexes are at risk of getting H. pylori infection, and suggest that pathologists should consider using immunohistochemistry in the evaluation of gastric biopsies. This may allow early detection and appropriate treatment, and hence decrease the risk of gastric cancer.