Helicobacter pylori infection and chronic gastritis; use of the Updated Sydney System in histopathological evaluation of gastritis

Objective Helicobacter pylori is a major cause for chronic gastritis and further it is associated with development of peptic ulcer disease and gastric cancer. Therefore, the objective of this study was to classify gastritis according to the updated Sydney system guidelines and nd the association of H. pylori with each of graded variable. Number of 152 dyspeptic patients who underwent upper gastro-intestinal endoscopy at a Teaching Hospital were enrolled. Of the 2 biopsies collected one was used for PCR to detect H. pylori. The other biopsy was xed in formalin followed by paran embedding and stained with H&E stain. Gastritis was classied microscopically according to the updated Sydney system. Results : Gastritis was reported over a wide age group ranging from 18-84 years with a mean age of 51 years. Based on histological ndings, 12% of patients were diagnosed as H. pylori associated chronic active gastritis. There was no signicant association between each graded variable and H. pylori positivity. Of the 152 dyspeptic patients 34 were positive by PCR for H. pylori infection. All the dyspeptic patients with H. pylori infection had chronic active gastritis, suggesting an etiologic role of the bacterium in the histologic lesion.

Based on histological ndings, 12% of patients were diagnosed as H. pylori associated chronic active gastritis. There was no signi cant association between each graded variable and H. pylori positivity. Of the 152 dyspeptic patients 34 were positive by PCR for H. pylori infection. All the dyspeptic patients with H. pylori infection had chronic active gastritis, suggesting an etiologic role of the bacterium in the histologic lesion.

Background
Helicobacter pylori is a Gram-negative, microaerophilic bacteria which colonizes the gastric epithelium inducing in ammation of the gastric mucosa [1,2]. It is a major etiological agent for chronic active gastritis and associated with gastric adenocarcinoma and mucosa associated lymphatic tissue lymphoma (MALToma) [2,3].
H. pylori has been classi ed as a class I carcinogen for humans by the International Agency for Research on Cancer (IARC) monograph committee in 1994. Prevalence of H. pylori infection is an enigma in Sri Lanka ranging from 3 to 70% [4,5,6]. Further resistance to clarithromycin is a problem [7] which results in eradication failure with triple therapy [6,8].
Sequence of in ammatory responses are induced in response to colonization of H. pylori and these responses coupled to cellular damage initiates a histological cascade (Normal mucosa > Chronic active gastritis > Gastric atrophy > Intestinal metaplasia > Dysplasia > Gastric cancer) towards the cancer [9].
The Sydney system has been introduced to grade and classify chronic gastritis in 1990. It has been updated in 1994 to provide a framework for a standardized description of chronic gastritis. In this updated version, ve histological variables namely chronic in ammation, neutrophil activity, glandular atrophy, intestinal metaplasia and Helicobacter pylori density are graded on a simple four point scale (absent or normal, mild, moderate and marked or severe abnormality) [10].
The aims of our study were to classify gastritis according to the updated Sydney system with graded variables and nd association of H. pylori with each of graded variables, as histo-pathological assessment of the gastric mucosa is known to be good predictor of cancer risk in H. pylori positive patient.

Methods
Hundred and fty two dyspeptic patients who underwent upper gastro intestinal endoscopy at Endoscopy unit at Colombo South Teaching Hospital, Sri Lanka were enrolled in this study. Ethical approval for the study was obtained from the Ethical Review Committee of University of Sri Jayewardenepura (723/13).
Two biopsy specimens were collected from the antrum during endoscopy of each patient after obtaining their written informed consent. Laboratory investigations were carried out at the Department of Microbiology and Department of Pathology, Faculty of Medical Sciences, University of Sri Jayewardanapura, Sri Lanka.
One biopsy was used for PCR targeting the glmM gene to identify H. pylori infection. DNA extraction from gastric biopsies and PCR was performed as mentioned in Ubhayawardana et al. 2015 [6].

Histological Investigations
The other specimen was xed in 10% formalin followed by para n embedding, sectioning and stained with H & E stain. H. pylori infection was diagnosed based on the typical appearance of the bacterium along the mucus layer covering the gastric mucous membrane.
Histopathological changes were examined and gastritis was classi ed microscopically according to the modi ed Sydney system [10]. Five main histological changes in gastric mucosa were graded (chronic in ammation, neutrophil activity, glandular atrophy, intestinal metaplasia and H. pylori density) by the same investigator (consultant histopathologist).

Results
The age of the study population ranged from 18-84 years with a mean age of 51 years. Majority of patients, 43% belonged to the age group above 55 years followed by 34% cases in 40-55 years age group and 23% in 18-39 age group. H. pylori infection was identi ed in 12% (18/152) by histology and in 22% (34/152) by PCR assay targeting glmM gene. Based on histological ndings, 12% (18/152) of patients were diagnosed as H. pylori associated chronic active gastritis, 82% (124/152) and 6% (10/152) of patients were diagnosed as having mild and moderate nonspeci c chronic gastritis respectively ( Figure   1).

Discussion
The nding of a high incidence of chronic gastritis among dyspeptic patients in this population is a matter of concern; therefore information on histological features of the gastric mucosa is important. Grading of gastritis conveys information on severity of mononuclear in ltration (In ammation) and polymorphonuclear cell in ltration (activity). Glandular atrophy and Intestinal metaplasia indicate the chronicity of the disease. These variables are also associated with increased risk of gastric cancer [11]. In the present study, 4% cases showed gastric atrophy and most of the cases were in 6 th and 8 th decades of their life, which suggest that it progresses as age advances in chronic gastritis patients. A previous study has shown that the prevalence of atrophic gastritis increased with increasing age [12].
Mononuclear in ltrate was more intense than the neutrophilic in ltrate in most biopsies. Higher histological scores for polymorphonuclear in ltrate and mononuclear in ltrate were observed in H. pylori infected patients. It has been reported that H. pylori causes continuous gastric in ammation in virtually all infected persons [13]. In a previous study, more intense polymorphonuclear cell in ltration was reported in patients with H. pylori associated gastritis and it was associated with duodenitis than in infected patients without duodenitis [14].
Major histopathological features such as gastric atrophy, Intestinal metaplasia were absent even in the patients who were moderately or severely infected with H. pylori. Similar results have been reported in a previous study done in Brazil in 2009 [15]. Risk of gastric cancer for these H. pylori positive patients with simple, non-atrophic gastritis may also be negligible since; intestinal metaplasia and atrophy are strongly associated with increased risk of gastric cancer.
H. pylori infection results in neutrophil activation and chronic gastritis; it has a role in the development of intestinal metaplasia [16]. However, early detection and eradication of H. pylori associated gastritis is important for patients in order to prevent the development of precancerous changes and gastric cancer.
Although, one of the most common causes for gastritis is colonization of gastric mucosa with H. pylori; cytomegalovirus infections, chronic idiopathic in ammatory and autoimmune disorders such as Crohn's disease and pernicious anemia, and chemical damage due to alcohol abuse or nonsteroidal antiin ammatory drug (NSAID) can be causes for gastritis in H. pylori negative dyspeptic patients [2]. In conclusion, all the dyspeptic patients with H. pylori infection had chronic active gastritis, supporting the etiologic role for the bacterium in the histologic lesion.

Limitations
In the study updated Sydney system was used for grading of gastritis. The updated Sydney system recommends gastric biopsies from ve different sites; however, this was not possible in practice in this study because of the ethical issues. Further clinicians do not agree to collect that many biopsies due to post biopsy bleeding risk for patients. Other limitations related with histology include higher cost, longer turnaround time and dependence on the skills of the operator.
Another limitation in the study was the methods of staining used, because some authors reported that staining with alcian blue-periodic acid Schiff (PAS) enhances the detection of incomplete intestinal metaplasia due to the detection of sulphated mucos substances [17]. This is an important limitation as it could prevent the detection of incomplete intestinal metaplasia. In the present study, a consultant pathologist performed the histological investigations and issued the report independently. Based on the ndings of the present study, it may be appropriate to conclude that for detection of H. pylori infection among symptomatic individuals it is necessary to use more than one diagnostic test as each test is associated with its own limitations. In this study, PCR has been shown to be a valuable method for detection of H. pylori in gastric biopsies.

Declarations
Ethics approval and consent to participate Ethical approval for the study was obtained from the Ethical Review Committee of University of Sri Jayewardenepura (No. 723/13). Two biopsy specimens were collected from the antrum during endoscopy of each patient after obtaining their written informed consent. Graded changes in gastric mucosa in H. pylori positive patients