Gastritis is an inflammatory condition of the gastric mucosa. Gastritis can be classified based on the underlying etiology i.e. Helicobacter pylori, non steroidal anti-inflammatory drugs, autoimmunity, bile reflux, allergic response and the histopathologic pattern (Srivastava and Lauwers, 2007).
Warren & Marshall in 1984 discovered that H. pylori are the most frequent causative agent of gastritis. H. pylori gastritis is a primary infection of the stomach and is the most common cause of chronic gastritis (Suzana et al., 2009). H. pylori are gram-negative rods that have the ability to colonize and infect the gastric mucosa. The bacteria survive within the mucous layer that covers the gastric surface epithelium and the upper portions of the gastric foveolae. This organism colonizes the gastric mucosa in a variety of ways: it is present free in mucus as well as surface adhesion, intercellularly and sometimes intracellularly. Once the organism has passed through the mucous layer, and has become established at the luminal surface of the stomach, an intense inflammatory response of the underlying tissue develops.
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It is estimated that half of the world's population is infected with H. pylori (BauerÂ andÂ Meyer, 2011). Infection withÂ H. pyloriÂ occurs worldwide, but the prevalence varies greatly among countries and among population groups within the same country (Suerbaum and Michetti, 2002). World over at least 50% of all people are infected, but an exact determination is not available, mostly because exact data are not available from developing countries (Salih, 2009).Â H. pylori infection is highly prevalent in Asia and in developing countries, and multifocal atrophic gastritis and gastric adenocarcinomas are more prevalent in these areas (Tanih et al., 2009). In the USA approximately 30-40% of adults are infected with H. pylori (Everhart et al, 2000). The prevalence of infection in minority groups and immigrants from developing countries is much higher (Mukherjee, 2012). Poor hygiene and crowded conditions may facilitate transmission of infection among the family members. The infection is related with poverty, limited education and residence in rural areas (Turner, 2010). In a large cross-sectional survey of adults in the United Kingdom, male gender, increasing age, shorter height, tobacco use, and lower socioeconomic status are all associated with positive H. pylori serology and the overall prevalence has been found to be 26% (Jackson et al., 2009).
In the developing countries like Pakistan, India, Bangladesh and Thailand, infection with H. pylori is more frequent among general population and is acquired at an early age (Singh and Ghoshal, 2006). According to a study carried out in Pakistan 84 percent patients with gastritis and 100 percent with duodenal ulcer had H. pylori infection (Shah et al., 2008). In Pakistan, H. pylori infection rate increases with advancement of age and lowering of socioeconomic status (Qureshi et al., 1999). A study in Pakistan revealed an early colonization/infection of infants with H. pylori and a prevalence of 67% at 9 months of age in a peri-urban community in Karachi (Nizami et al., 2005). In another study done at Karachi the overall prevalence of H. pylori is 87.03% and in chronic gastritis it is 66.66 % (Asif et al., 2011).
H. pylori infection is not only linked to chronic gastritis but
also to incomplete and rarely complete intestinal metaplasia (Khan et al., 2003), peptic ulcer, adenocarcinoma (Schneller et al., 2006) and gastric lymphoma arising from mucosa associated lymphoid tissue (MALT) (Kumar et al., 2006). H. pylori in its first colonization cause an acute superficial gastritis including neutrophilic infiltration between surface and foveolar epithelial cells and within gastric pits. The surface epithelium shows degenerative changes with loss of mucin and increased exfoliation and the lamina propria is edematous (Suzana et al., 2009). Histological changes of chronic gastritis include lymphocytes, plasma cells infiltration and other inflammatory cells in lamina propria (Alireza et al., 2008). Neutrophilic infiltration is seen in active cases of disease. Lymphoid aggregate with germinal centre can also be observed. Chronic H. pylori gastritis exhibits more prominent gastritis in the antrum than in the corpus (Dixon et al., 1996).
In disease of longer duration, gastric atrophy and intestinal metaplasia are observed (Guarner et al., 2001). According to the data in the West, atrophy and intestinal metaplasia are premalignant conditions seen in patients with H. pylori associated chronic gastritis (Micu et al., 2010). With expansion of intestinal metaplasia, the number of H. pylori organisms that are detectable in the stomach decreases because H. pylori are excluded from areas of metaplastic epithelium (Mukherjee et al., 2010). The prevalence of atrophy in Dutch patients is 42% and in Chinese patients is 52%. The prevelance of intestinal metaplasia in China is 26% and in Denmark is 32 % (Chen et al., 2001). In the United Arab Republic (high incidence of gastric cancer), the prevalence of atrophy is 26%, and intestinal metaplasia is 11 % (Zaitoun, 1994). Significant improvements in gastricÂ atrophyÂ and intestinal metaplasia after H. pylori eradication may decrease the risk of gastric cancer (Khodama et al., 2012). Intestinal type gastric cancer may be the end stage in a progression from simple gastritis to gastric atrophy, metaplasia, dysplasia and carcinoma. The crucial step is in these events is the development of atrophy (Wong et al., 2004). The removal ofÂ H. pyloriÂ before the development of atrophy appears to prevent carcinogenesis (Wong et al., 2004). Thus, the main role of H. pyloriÂ in intestinal type gastric cancer appears to be the induction of atrophy, implying that research should concentrate on this process. The H. pylori screening of population and treatment has the potential of dramatically reducing global gastric cancer mortality (Moayyedi and Hunt, 2004). Moreover, the bacterial density has been correlated with gastric inflammation (Gallo et al., 2003). A study carried out at Civil Hospital, Karachi shows frequency of H. pyloriÂ in biopsy proven gastritis and its association with lymphoid follicle formation (Siddiqui et al., 2011).
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The Sydney System is a novel classification and grading of gastritis that was devised by a group of experts at the 9th World Congress of Gastroenterology in Sydney, Australia in 1990. Experts emphasized the importance of combining topographical, morphological and etiological information for the diagnostic evaluation of gastritis. In 1994 in Houston, Texas, experts devised the new Updated Sydney System (Dixon et al., 1996; Grieken et al., 2001). The reproducibility of grading H. pylori related gastritis is high using the Updated Sydney System. Therefore, a system which could sustain good reproducibility in reporting these observations is very important (Amoueian et al., 2008). The Updated Sydney System has a scale of 0-3 for scoring the features of chronic gastritis (Dixon et al., 1996). In order to improve assessment of minor degrees of alteration, a detailed histopathological classification can be used, which also provides numerical data for statistical analysis. We did not find any local literature supporting the significant association of H. pylori load or density with severity of atrophy, intestinal metaplasia, activity or degree of chronic inflammatory infiltrate. In order to address this issue the present study was designed.
AIMS AND OBJECTIVES
The aims and objective of this study in patients of chronic gastritis were to: (a) Determine the density of H. pylori.
(b) Determine the degree of inflammatory activity, chronic inflammatory infiltrate, atrophy and intestinal metaplasia semi quantitatively.
(c) Explore the association of density of H. pylori with degree of:
Chronic inflammatory infiltrate
REVIEW OF LITERATURE
2.1 ANATOMY OF THE STOMACH
2.1.1 Gross Anatomy (Snell, 2012).
The stomach is situated in the upper part of the abdomen, extending from beneath the left costal margin region into the epigastric and umbilical regions. Much of the stomach lies under cover of the lower ribs. It is roughly J-shaped and has two openings, the cardiac and pyloric orifices; two curvatures, the greater and lesser curvatures; and two surfaces, an anterior and a posterior surface. The stomach is divided into the following parts:
Fundus is dome-shaped and projects upward and to the left of the cardiac orifice. It is usually full of gas. Body extends from the level of the cardiac orifice to the level of the incisura angularis, a constant notch in the lower part of the lesser curvature. Pyloric antrum extends from the incisura angularis to the pylorus. Pylorus is the most tubular part of the stomach. The thick muscular wall is called the pyloric sphincter, and the cavity of the pylorus is the pyloric canal.
The stomach has two curvatures, the lesser and the greater curvature. The cardiac orifice is where the esophagus enters the stomach. The pyloric orifice is formed by the pyloric canal, which is about 1 in. (2.5 cm) long.
The arteries are derived from the branches of the celiac artery. The left gastric artery arises from the celiac artery. It supplies the lower third of the esophagus and the upper right part of the stomach. The right gastric artery arises from the hepatic artery. It supplies the lower right part of the stomach. The short gastric arteries arise from the splenic artery at the hilum of the spleen and pass forward in the gastrosplenic omentum (ligament) to supply the fundus. The left gastroepiploic artery arises from the splenic artery at the hilum of the spleen and passes forward in the gastrosplenic omentum (ligament) to supply the stomach along the upper part of the greater curvature. The right gastroepiploic artery arises from the gastroduodenal branch of the hepatic artery. It supplies the stomach along the lower part of the greater curvature.
2.1.2 Histology (Ross and Pawlina, 2011)
The wall of the stomach is composed of four layers i.e. mucosa, submucosa, muscularis propria and serosa. The mucosa is thrown into prominent folds and consists of gastric glands that extend from the level of muscularis mucosae to open into stomach lumen via gastric pits or foveoli. The submucosa is loose and distensible and contains larger blood vessels. The muscularis propria is composed of inner oblique, middle circular and outer longitudinal layers. The serosal surface covers the peritoneal surface.
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The different types of cells present in gastric mucosa are: Surface mucous cells which line the luminal surface of the stomach and gastric glands. Neck mucous cells are present in the neck and base of glands. Parietal or oxyntic cells are distributed throughout the length of the gland, but numerous in the middle portion. These areÂ large, rounded cells with eosinophilic cytoplasm and centrally located nucleus which produce gastric acid. Chief, peptic or zymogenic cells are clustered at the base of the gland and are identified by basally located nuclei and strongly basophilic granular cytoplasm. These are the pepsin secreting cells. Stem cells are found mainly in the neck of the gastric glands. These undifferentiated cells divide continuously to replace all other cell types. Neuroendocrine cells are part of diffuse neuroendocrine system and are present at the base of glands.
The gastric mucosa consists of surface epithelium, gastric pits and gastric glands. The gastric glands extend from the muscular mucosae to extend into the stomach lumen via gastric pits. The foveolar cells lining the surface and gastric pits are identical throughout the stomach. The gastric glands vary in different regions of stomach. Gastric pits occupy approximately 25% of the mucosa. Pits lie parallel to one another. These are separated by the lamina propria. There is more lamina propria separating the pits than between the glands. In normal gastric biopsy degree ofÂ pit and glandular separation should be the same through out the biopsy.
Cardia is the small area of predominantly mucus secreting glands surrounding the entrance of the esophagus. Glands are less coiled than in the antral glands. The pits are shorter than the antropyloric pits. The fundus or body consists of straight, tubular glands. Strands of muscularis mucosae extend between the glands from the base. The glands secrete gastric juices as well as protective mucus so parietal and chief cells are numerous in fundic glands. Pylorus or antrum, the glands here are branched and open into deep irregular shaped pits. The glands are composed of mucus secreting cells. Mucus secreted by pyloric glands lubricates and protects entrance to the duodenum. Scattered 'G' cells (endocrine cells), secrete gastrin.
2.2 PHYSIOLOGY OF STOMACH
The motor functions of the stomach are threefold:
(1) Storage of large quantities of food until the food can be processed in the stomach, duodenum, and lower intestinal tract.
(2) Mixing of this food with gastric secretions until it forms a semiï¬‚uid mixture called chyme
(3) Slow emptying of the chyme from the stomach into the small intestine at a rate suitable for proper digestion and absorption by the small intestine (Hall, 2011).
2.3 HELICOBACTER PYLORI
H. pyloriÂ are gram-negative bacteria that infect the stomach. It is the cause of gastric inflammation which is strongly linked to the development of gastric or duodenal ulcers. H. pylori survive in the highly acidic pH of the stomach by secreting high amounts of urease enzyme which serves as its protective covering. It is also a highly variable bacterium; even in a single infected patient, all the bacteria are not identical due to its independent adaptations to the changing conditions in the stomach (Castillo, 2010).
During the 1970s, there was a sudden increase in the incidence of peptic ulcer disease and gastritis. In the year 1979, due to his interest in the new gastric biopsies, Warren who was a biologist, noticed small curved bacteria growing on the surface of 50% of the gastric biopsies taken. These gastric biopsies were taken from the antrum of the stomach of the patients. He also noted signs of inflammation in the area where the bacteria were seen.
Over the next 2 years, he painstakingly gathered numerous examples of gastric biopsies with the bacteria and showed that it usually occurs with chronic gastritis. In 1981, Warren met Marshall who was a clinician to talk about the newly found curved bacteria from the gastric biopsies. Together, the two
found that the bacteria present in almost all patients with gastritis, duodenal ulcer or gastric ulcer. The Nobel prize in physiology or medicine of 2005 was awarded to Marshall and Warren for their discovery of bacterium H. pylori and its role in the development of gastritis and peptic ulcer disease in humans (Castillo, 2010).
H.Â pylori are motile, with a rapid cork-screw-like or slower wave-like motion due to flagellar activity. Strains of most species have bundles of multiple sheathed flagella with a polar or bipolar distribution. Other species have only a single polar or bipolar flagellum (Solnick and Vandamme, 2001).
H. pylori are non spore forming gram negative bacteria. The cellular morphology may be curved, spiral, or fusiform, typically 0.2 to 1.2 Î¼m in diameter and 1.5 to 10.0 Î¼m long. The spiral wavelength may vary with the age, the growth conditions, and the species identity of the cells. In old cultures or those exposed to air, cells may become coccoid.
2.4 PATHOPHSIOLOGY OF H. PYLORI GASTRITIS
The disease most often presents as most predominantly antral gastritis with high acid production, although the levels of gastrin are low. In some patients the gastritis progresses to involve the body or fundus. This pangastritis is associated with multifocal mucosal atrophy, reduced acid secretion, intestinal metaplasia, and increased risk of gastric adenocarcinoma.
H. pylori organisms have adapted to the ecologic niche provided by gastric mucus. Although H. pylori may invade the gastric mucosa, this is not evident histologically and the contribution of invasion to disease is not known. Four features are linked to H. pylori virulence. These are as follows:
Flagella, which allow the bacteria to be motile in viscous mucus.
Urease, which generates ammonia from endogenous urea and thereby elevates local gastric pH.
Adhesins, which enhance their bacterial adherence to surface foveolar cells.
Toxins, such as cytotoxin-associated gene A (Cag A).that may be involved in ulcer or cancer development by poorly defined mechanisms (Turner, 2010).
2.4.1 Interaction between host and H. pylori
The chronic antral gastritis may progress to pangastritis, resulting in multifocal atrophic gastritis. The interaction between host and H. pylori seems to play an important role. Particular polymorphisms in the gene encoding pro inflammatory cytokine interleukin-1B (IL-1B) and tumor necrosis factor (TNF) influence the outcome in H. pylori infection. Severity of the disease may be influenced by genetic variation among H. pylori. For example, the CagA gene, a marker of pathogenicity is present in 90% of H. pylori isolates found in populations with elevated gastric cancer risk (Turner, 2010).
2.4.2 Patterns of H. pylori gastritis
In the majority of infected individuals, H. pylori gastritis is to some degree more marked in the antrum than in the corpus. When there is a substantial difference between the two compartments, such that there is minimal inflammation in the corpus and marked involvement of the antrum, the gastritis is designated "antral predominant." This pattern is found in patients with duodenal and prepyloric ulceration (Dixon, 2001).
If the H. pylori colonize the antrum of the stomach, the inflammatory response of the G cells in the site is to secrete more gastrin. The increase in gastrin will then trigger the parietal cells in the corpus of the stomach to produce more gastric acids. Increase in acids damages the duodenum and ulcerations may occur. On the other hand, if the H. pylori colonize the corpus of the stomach where the acid secreting cells called parietal cells are located, there will be a marked decrease in acid production and secretion which will eventually cause atrophy of the stomach lining which may lead to gastric ulcers (Castillo, 2010).
2.5 MORPHOLOGICAL CHANGES SEEN IN H. PYLORI ASSOCIATED CHRONIC GASTRITIS
The initial, acute phase of infection is subclinical in the great majority of subjects. Following ingestion, organisms penetrate through the viscid mucous layer and multiply in close proximity to the surface epithelial cells. The epithelium responds to infection by mucin depletion, cellular exfoliation, and compensatory regenerative changes. Neutrophils infiltrate into foveolar and surface epithelium, and lamina propria edema is conspicuous. Collections of neutrophils in the foveolae or glands (pit abscesses) and adherent neutrophilic exudate on the surface may also be present. The acute phase is short lived. In a small minority of people, and particularly in childhood, the organisms may be spontaneously cleared, the polymorph infiltrate resolves, and appearances return to normal. In the majority, however, the host immune response fails to eliminate the infection and over the next 3 or 4 weeks there is a gradual accumulation of chronic inflammatory cells that come to dominate the histological picture. As a consequence, the diagnosis of an acute neutrophilic gastritis gives way to that of an active chronic gastritis (Sobala et al., 1991).
2.5.2 Chronic inflammatory infiltrate
Diffuse chronic H. pylori gastritis predominantly affects the pyloric mucosa. Although the inflammatory changes are scanty in the corpus, organisms may be found throughout the surface mucus. The mucosa shows a dense inflammatory infiltrate, in which plasma cells are prominent. In the corpus any inflammation present is confined to superficial zone. In the antral mucosa, the infiltrate is predominantly superficial,but can involve the whole thickness and may separate the glands without causing atrophy. Lymphoid follicles with germinal centers are usually seen in deeper portions of mucosa. This finding is pathognomonic for presence of H. pylori. The surface and foveolar epithelium is infiltrated by neutrophils, which may be so prominent that pit abscesses are formed. This neutrophil infiltration is called active gastritis and is seen predominantly in those areas where H. pylori are most abundant (Owen, 2010).
Atrophy in the stomach is conventionally defined as loss of glandular tissue from repeated or continuing mucosal injury and is a common denominator in all pathological processes causing progressive mucosal damage, including longstandingÂ H. pylori infection Thus, loss of glands may follow erosion or ulceration of the mucosa, with destruction of the glandular layer, or as a result of a prolonged inflammatory process in which individual glands undergo destruction. When such loss occurs, it is followed by fibrous replacement. However, atrophy can also be thought of as simply "a loss of specialized or functional cells." Under this broader definition it is possible to include situations in which there is loss or replacement of parietal and chief cells without glandular destruction (Dixon, 2001).
The prevalence ofÂ H. pylori positivity declines with increasing glandular atrophy. There are two main reasons for the loss of organisms. First,Â H. pyloriÂ only colonizes gastric epithelium; thus, the organisms are absent from areas of complete intestinal metaplasia. Second, the organism only thrives within the narrow pH range provided by a partially acidic environment and the hypochlorhydric stomach is inimical toÂ H. pyloriÂ (Clyne et al., 1995). Therefore, the failure to demonstrateÂ H. pyloriÂ in the atrophic stomach does not deny a role for infection in the causation of the underlying gastritis.
2.5.4 Intestinal metaplasia
Metaplasia is defined as "a potentially reversible change in which a fully differentiated cell type is replaced by another differentiated cell type, and usually represents a change to cells better able to withstand an adverse environment". Thus, intestinal metaplasia represents a change from a gastric epithelial phenotype to a small- or large-intestinal phenotype. Metaplasia is always associated with some abnormal stimulation of growth, for example, during regeneration following mucosal injury (Mukawa et al., 1987; Oohara et al., 1983).
Intestinal metaplasia is a common finding in chronic gastritis of all causes. Â Its extent usually, but not invariably, parallels the development of atrophy. Intestinal metaplasia is found more frequently inÂ H. pylori positive than negative cases (Dixon, 2001).
2.5.5 Lymphoid follicles
The normal gastric mucosa contains very few lymphocytes in the lamina propria. Lymphoid follicles and aggregates are characteristic of H. pylori associated gastritis (Chen et al., 2002). H. pylori infection causes an immunological response, leading to chronic gastritis with formation of lymphoid follicles within the stomach. These lymphoid follicles resemble nodal tissues found throughout the body and are composed of reactive T cells and activated plasma cells and B cells (Ahmad et al., 2003).
MATERIALS AND METHODS
This study was conducted in the Department of Pathology, Army Medical College, National University of Sciences and Technology and Military Hospital Rawalpindi, Pakistan.
3.2 DURATION OF STUDY
December 2011 to November 2012
3.3 SAMPLE SIZE
In this study 100 biopsies of chronic gastritis patients were included.
3.4 SAMPLING TECHNIQUE
Non probability convenience sampling technique was used.
3.5 SAMPLE SELECTION
Gastric antral biopsies of chronic gastritis patients of all ages and both sexes were included in the study.
3.5.2 Exclusion criteria:
Gastric biopsies of patients who were receiving or had received H. pylori eradication treatment were not included in the study.
3.6 STUDY DESIGN
A cross-sectional correlational study
3.7 PATIENTS'DATA AND EXPERIMENTAL PROTOCOL
Permission of the ethical committee was taken. Biopsies of 100 dyspeptic patients having H. pylori gastritis were enrolled prospectively into the study. The specimens were mostly received from the Gastroenterology unit of Military Hospital, Rawalpindi. The patients were both serving/ retired military personnel as well as civilians living in Rawalpindi and its suburbs. Patients of all ages and both sexes having undergone gastric biopsy were included in the study. Specimens were taken as a whole in 10% formal saline and were received in the Pathology Laboratory, Army Medical College, Rawalpindi. Sample of each patient were given a laboratory number and record was maintained.
3.7.1 Collection of clinical data
The relevant clinical information and demographic data was obtained from the laboratory request form. Clinical data was collected prospectively from all study participants in cases where it was not mentioned on request form. Data included the following information: results of endoscopic investigations, age, sex, symptoms, concomitant medication (in particular intake of proton pump inhibitors (PPI), antibiotics or bismuth salts, non steroidal anti inflammatory drugs). History regarding relevant previous and concomitant diseases including abdominal surgery was taken. All the data was entered in patient's proforma (Annexure I).
3.8 TISSUE PROCESSING
3.8.1 Specimen Processing
The tissue was taken as whole on filter paper and enclosed in properly labeled plastic cassettes with perforated walls and was placed in LEICA TP 1020 (Germany) automatic tissue processor. The following processing schedule was followed.
Dehydration was done in ascending series of alcohol
80% alcohol 1 hour
95% alcohol 1 hour
Absolute alcohol - I 1 hour
Absolute alcohol - II 1 hour
Two changes of xylene were used.
Xylene - I 2 hours
Xylene -II 2 hours
Paraffin with melting point 56-58 c was used for this purpose.
First change 2 hours
Second change 2 hours
Paraffin embedding centre (LEICA EG 1160- Germany) was use for this purpose. Filtered paraffin with melting point 56-58 c was used for embedding. Blocks were made using plastic cassettes. Each cassette was filled with molten paraffin wax and tissue was placed in the center of the cassette. The cassette was then allowed to cool on the cold plate of the paraffin embedding centre.
Paraffin blocks were placed in block holder of rotary microtome, LEICA RM 22.5 (Germany), and 3-4 um thick sections were made. Sections were floated in warm water bath at 45 c and were taken separately on 2 different slides, which were albumin coated. Slides were further dipped horizontally in 70% alcohol bath to remove any creases. The slides were kept in a slanting position for about half an hour to drain excess of water. The section was then dried on hot oven at 60c for about 15-30 minutes.
Following stain was used in LEICA autostainer (Germany) XL.
Haematoxylin and Eosin (H&E) stain for routine histology (Appendix II).
Giemsa stain was used for demonstration of H. pylori (Appendix III).
3.9 MICROSCOPIC EXAMINATION
The gastric biopsies were scored semi quantitatively according to Updated Sydney System (Dixon et al., 1996). The Updated Sydney System has a scale of 0-3 for scoring the features of chronic gastritis. In order to improve assessment of minor degrees of alteration, a detailed histopathological classification was used, which also provided numerical data for statistical analysis (Chen et al., 1999). The following histopathological parameters were examined on each slide: density of H. pylori, inflammatory activity, chronic inflammation, atrophy and intestinal metaplasia. Each category (mild, moderate, and severe) was divided into two subcategories, resulting in a score on a scale of 0-6 (none, 0; mild, 1-2; moderate, 3-4; severe, 5-6). According to this classification, the histopathological parameters were graded as follows:
3.9.1 Density of H. pylori colonization:
The density of H. pylori was graded as follows:
1: H. pylori found only in one place after a careful search
2: only a few H. pylori found
3: scattered H. pylori found in separate foci
4: numerous H. pylori in separate foci
5: nearly complete gastric surface covered by layer of H. pylori
6: continuous gastric surface covered by a thick layer of H. pylori
3.9.2 Degree of inflammatory activity:
The degree of inflammatory activity was graded according to the density of neutrophils in the gastric mucosa:
1: only one crypt involved per biopsy
2: two crypts involved per biopsy
3: many crypts(up to 25% )involved per biopsy
4: 25-50% of crypts involved per biopsy
5: more than 50% crypts involved per biopsy
6: all crypts involved
3.9.3 Degree of chronic inflammatory infiltrate:
The degree of chronic inflammatory infiltrate was graded as follows:
1: scattered chronic inflammatory cells<10 cells / high power field
2: scattered chronic inflammatory cells>10 cells /high power field
3: some areas with dense chronic inflammatory cells
4: diffuse infiltration with dense chronic inflammatory cells
5: nearly the whole mucosa contains dense infiltrate which separates gastric glands
6: entire mucosa contains a dense chronic inflammatory cell infiltrate
3.9.4 Degree of atrophy:
Atrophy was graded as follows:
1: foci where a few gastric glands are lost or replaced by intestinal type of epithelium
2: small areas in which gastric glands are lost or replaced by intestinal type of epithelium
3: up to 25% gastric glands lost or replaced by intestinal type of epithelium
4: 25-50% of gastric glands lost or replaced by intestinal type of epithelium
5: more than 50% of gastric glands lost or replaced by intestinal type of epithelium
6: only a few small areas of gastric glands remaining
3.9.5 Degree of intestinal metaplasia:
The degree of intestinal metaplasia was graded according to the extent of glandular tissue replaced by intestinal type of epithelium:
1: only one focus (one crypt) replaced by intestinal epithelium
2: one focal area(1-4 crypts) replaced by intestinal epithelium
3: two separate foci
4: multiple foci in the biopsy
5: >50% epithelium replaced by intestinal epithelium
6: only a small area of gastric epithelium is not replaced by intestinal metaplasia
Non quantitative variables
Non quantitative histopathological features like lymphoid aggregates, ulcer slough, superficial epithelial damage and nuclear reactive changes were not graded, but simply assessed in case of their presence or absence. Dysplasia was also assessed as an important factor in the histological sequence leading to gastric cancer.
3.10 DATA ANALYSIS PROCEDURE:
All data was collected through specifically designed proforma (Appendix I). Findings were entered and analyzed through SPSS version 17. Quantitative variables were represented using frequency and percentages. Spearman's rank correlation test was used for calculating relationship between H. pylori density and other variables. The experimental findings were considered statistically significant if p value was less than 0.05 (p<0.05).
3.10.1 Spearman's Rank Order Correlation using SPSS
The Spearman's rank-order correlation is the nonparametric version of theÂ Pearson product-moment correlation. Spearman's correlation coefficient measures the strength and direction of association between two ranked variables measured on an ordinal scale. It is denoted by the symbolÂ rsÂ (or the greek letterhttps://statistics.laerd.com/statistical-guides/img/spearman-rho.jpg, pronounced rho) and measures the tendency of Y to increase or decrease as X increases. The test is used for either ordinal variables or for interval data that has failed the assumptions necessary for conducting the Pearson's product-moment correlation.
3.10.2 Interpretation of Spearman's Rank Order Correlation CoefficientÂ rsÂ
Spearman's Rank Order Correlation Coefficient rsÂ
Strength of Association
0.1 to 0.5
-0.1 to -0.5
0.5 to 0.75
-0.5 to -0.75
0.75 to 1.0
-0.75 to -1.0