Helicobacter pylori-Associated Infection: A Comprehensive Histopathological Analysis of Gastric Biopsies from Patients of Pakistan

Abstract

Helicobacter pylori is a gastric pathogen that induces chronic gastritis, which may progress to neutrophilic activity, glandular atrophy, intestinal metaplasia, and gastric carcinoma. The aim of this study was to evaluate H. pylori-induced tissue damage. A total of 602 gastric biopsy samples were collected, categorized, and analyzed using hematoxylin and eosin and Giemsa staining, followed by molecular confirmation through PCR targeting the species-specific 16S rRNA gene. H. pylori density and histopathological features were evaluated and graded according to the updated Sydney classification system. H. pylori was detected in 55% (n = 334) of cases, and the antrum (50.83%, p < 0.00001) was the predominant site. A slightly higher prevalence was observed in females, accounting for 56.9% compared to males at 43.1%, which was attributed to sociocultural exposure differences. Individuals aged 11–40 years accounted for 58.3% (n = 195), highlighting early-age acquisition of infection. H. pylori infection was significantly linked to moderate-to-severe inflammation (63.2%, p < 0.00001) and neutrophilic activity (53.3%, p < 0.00001). Intestinal metaplasia and atrophy were infrequent, present in 0.6% (95% CI, 0.02, p = 0.149) and 0.9% (95% CI, 0.05, p = 0.430) of individuals. H. pylori infection causes chronic inflammation and neutrophilic infiltration of the stomach mucosa. Early identification and histopathological examination are essential in assessing H. pylori-related gastric pathology.

1. Introduction

Helicobacter pylori, a spiral-shaped, Gram-negative, microaerophilic bacterium, thrives in the hidden folds of the human stomach, beneath the thick layer of protective mucus [1]. Its characteristic corkscrew motility, mediated by multiple flagella, enables penetration of the viscous mucus layer, while urease production neutralizes gastric acid by hydrolyzing urea to ammonia, creating a microenvironment to survive in hostile acidic conditions [2]. Clinically, H. pylori presents a wide spectrum, from asymptomatic colonization to chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and adenocarcinoma [3]. Transmission primarily occurs via the oral–oral and fecal–oral routes in childhood, with socioeconomic and hygiene factors driving higher prevalence in developing regions [4]. Diagnosis relies on invasive methods (biopsy for histopathology, rapid urease test, culture) and non-invasive tests (urea breath test and stool antigen detection). The most revealing insight into H. pylori pathogenesis comes not just from detection but from a direct histological examination of the tissue it transforms [5]. Histopathological examination remains the gold standard for confirming and assessing mucosal damage [6].

Gastritis, the most common and earliest pathological complication of H. pylori, is defined as inflammation of the gastric mucosa. It can histologically be categorized as acute or chronic. Acute gastritis is marked by a rapid influx of neutrophils into the lamina propria and epithelium, often triggered by infection, non-steroidal anti-inflammatory drugs, or alcohol. Chronic gastritis features sustained mononuclear infiltration, predominantly lymphocytes and plasma cells. Persistent neutrophilic and lymphoplasmacytic inflammation injures the gastric epithelium, leading to glandular loss termed as atrophy [7]. Over time, atrophic mucosa is replaced by intestinal-type epithelium termed ‘intestinal metaplasia’. This process is driven by chronic oxidative stress and cytokine-mediated signaling [8]. Metaplastic epithelium accumulates genetic and epigenetic alterations such as DNA methylation of tumor suppressor genes and activation of oncogenic pathways from progress through dysplasia to invasive adenocarcinoma [9]. To standardize the evaluation of these histological patterns, the updated Sydney classification system was developed, which grades gastritis based on five key features: chronic inflammation, neutrophilic activity, atrophy, intestinal metaplasia, and density of H. pylori [10].

In low–middle-income countries like Pakistan, the prevalence remains high (58–65%) due to suboptimal sanitation, overcrowding, and lack of screening. Recent global data [11,12] underscore regional variability and emerging antibiotic resistance. The present study investigates the correlation between H. pylori presence and the full spectrum of histopathological changes graded by the updated Sydney classification system and the significance of combined morphological and molecular diagnostic approaches. By analyzing the prevalence and severity of inflammation, neutrophilic activity, atrophy, and intestinal metaplasia in individuals infected with H. pylori, this study aims to illuminate the pathological footprints of H. pylori.

2. Materials and Methods

2.1. Sampling

This cross-sectional study was conducted at the LabCorp Diagnostic and Research Centre, Lahore, Pakistan. A total of 602 gastric biopsies were included in the study undertaken. The study was approved by the ethical committee of LabCorp Diagnostic and Research Centre, Lahore, Pakistan (LABCORP/EC/2022/053 dated 5 May 2022). The data was obtained through a questionnaire, and informed written consent was taken from the patients.

2.2. Preparation of Gastric Biopsy Samples for Staining

The gastric biopsy samples were initially fixed in 10% neutral-buffered formalin to preserve cellular and tissue architecture effectively [11]. Grossing was performed in accordance with international guidelines for gastrointestinal histopathology, such as the updated Sydney classification system, which ensures standardized assessment of gastric inflammation. Following grossing, tissue processing was carried out using an automated tissue processor (Thermo Fischer Scientific, Vilnius, Lithuania), and specimens were embedded on an embedding station (Leica EG1150 C, Heidelberger, German) in paraffin wax to facilitate sectioning. Thin sections of 2–4 µm were obtained using a rotary microtome (Epredia HM 325, Epredia, Portsmouth, NH, USA). Following microtomy, 2–4 µm thick paraffin-embedded gastric tissue sections were mounted onto clean glass slides for downstream staining procedures.

2.3. Hematoxylin and Eosin (H&E) Staining for H. pylori Detection and Histopathological Details

The mounted biopsy glass slides were processed for hematoxylin and eosin staining (MilliporeSigma, Burlington, VT, USA) to assess H. pylori presence and histopathological changes. Briefly, the H&E staining was performed according to protocol as described earlier [12]. The slides were deparaffinized in three changes in xylene (5 min each), followed by rehydration through descending grades of ethanol (100%, 95%, 80%, 70%) and rinsed in distilled water. The sections were then immersed in hematoxylin for 5–8 min to stain cell nuclei. The sections were rinsed in running tap water, and 1% ammonia water was added to enhance nuclear detail. After rinsing, the sections were counterstained with eosin-Y (1%) for 1–2 min to stain cytoplasmic components. The slides were dehydrated through ascending grades of ethanol and cleared in xylene (Thermo Fischer Scientific, Vilnius, Lithuania). After clearing the step, the slide was mounted with Distyrene, Plasticizer, and Xylene (DPX) (Merck, Darmstadt, Germany).

2.4. Giemsa Staining to Evaluate the Presence of H. pylori

Giemsa staining (Merck, Darmstadt, Germany) was used to highlight H. pylori within the gastric mucosa, following protocols optimized for microbial detection [13]. After deparaffinization and rehydration, sections were stained in freshly prepared Giemsa working solution (1:5 dilution in pH 6.8 buffered distilled water) for 10 min at room temperature. Slides were then dehydrated, cleared in xylene, and mounted using Distyrene, Plasticizer, and Xylene (DPX).

2.5. Microscopic Examination of H&E and Giemsa-Stained Slides

The stained slides were independently evaluated under a light microscope (OLYMPUS CX23, Evident, Tokyo, Japan) by an experienced histopathologist for definitive diagnosis. Histopathological assessment included evaluation of H. pylori density, type and degree of gastric inflammation, activity, atrophy and intestinal metaplasia as per the standard protocol adopted by the Updated Sydney Classification System.

2.6. Detection of H. pylori in Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Using Species-Specific 16S rRNA Gene Amplification

The FFPE tissue blocks were sectioned into six serial slices each of 5 µm thickness using a microtome [14]. Deparaffinization was carried out by adding 1 mL xylene to each sample, followed by incubation at 50 °C for 10 min [14]. This step was repeated twice to ensure complete removal of paraffin. The tissue was then subjected to ethanol and washed twice with 100% ethanol, each followed by centrifugation and decanting of the supernatant. Following dehydration, samples were air-dried at room temperature for 1 h to allow residual ethanol to evaporate. For tissue digestion, 250 µL of digestion buffer and 20 µL of proteinase-K were added to each centrifuge tube. The samples were then incubated at 56 °C for 24 h to ensure complete lysis of the tissue matrix. DNA extraction was subsequently performed using GeneJET FFPE according to the manufacturer’s protocol (Thermo Fisher Scientific, Vilnius, Lithuania). The final elution volume for purified DNA was 50 µL. Extracted DNA was stored at −20 °C until further use. A species-specific detection of H. pylori was performed by PCR targeting the 16S rRNA gene (138 bp, forward: 5′-GCGACCTGCTGGAACATTAC-3′, reverse: 5′-CGTTAGCTGCATTACTGGAG-3′ [15]. The PCR was carried out in a 25 µL reaction mixture containing 12 µL of 2X PCR master mix (Thermo Fischer Scientific, Vilnius, Lithuania), 1 µL of 10 µm forward and reverse primers each, 2 µL of template DNA, and 8.5 µL of nuclease-free water. The thermal cycling included an initial denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide and imaged under UV light. A 50 bp ladder with positive and negative controls was added in each run.

2.7. Statistical Analysis

All the experiments were repeated three times. Fisher’s exact test was used for a 2 × 2 contingency table (https://www.socscistatistics.com/tests/fisher/default2.aspx, accessed on 10 August 2025). Fisher’s exact test (Freeman–Halton extension) (https://www.danielsoper.com/statcalc/calculator.aspx?id=58, accessed on 11 August 2025) was used for a 2 × 3 contingency table. A p-value of ≤0.05 was considered significant.

3. Results

3.1. Presumptive Presence of H. pylori on H&E and Giemsa Staining

The H&E-stained biopsies under light microscopy showed pink-colored, spiral-shaped rods, which were presumptive H. pylori (Figure 1a). The microscopic examination of Giemsa-stained tissue showed dark blue, curved, or spiral-shaped bacilli localized at the epithelial surface or within the mucosal glands, contrasting against a pink cytoplasmic background (Figure 1b). Overall, on Giemsa staining, H. pylori presence was observed in 334 (55%) gastric tissue samples out of 602 samples.

3.2. Molecular Detection of H. pylori

The presumptive H. pylori-positive Giemsa-stained samples were processed for PCR detection of H. pylori. An amplified product of 138 bp exhibited amplification of the H. pylori-specific 16S RNA gene (Figure 2). PCR amplification confirmed the presence of H. pylori in all Giemsa-positive cases (n = 334).

3.3. Tissue Detail of Gastric Biopsies on H&E Staining

The tissue detail of gastric biopsies was investigated using H&E staining. Gastric antral biopsy with mild chronic gastritis and moderate chronic gastritis with lymphoid follicles was observed. The H&E-stained biopsies under light microscopy showed cell nuclei appeared blue to purple, while cytoplasm and connective tissue structures exhibited shades of pink (Figure 3).

3.4. Distribution of H. pylori with Respect to Density and Site of Infection

Based on bacterial density, 194 (32%) exhibited few organisms, 108 (18%) showed moderate density, and 32 (5%) demonstrated heavy colonization. H. pylori was absent in 268 (45%) of cases. Among the 334 H. pylori-positive gastric biopsy samples, infection was significantly localized in the antrum, accounting for 50.83% (p < 0.00001) of cases, followed by combined antrum and body involvement at 4.32% of cases, while isolated body involvement was rare at 0.33% of cases (

Table 1. Distribution of H. pylori with respect to density and site of infection.

Density n (%)				Site n (%)
				Antrum	Body	Antrum and Body	p-Value
Absent	Few	Moderate	Many	Present	Present	Present
268 (45)	194 (32)	108 (18)	32 (5)	306 (50.83)	2 (0.33)	26 (4.32)	<0.00001

Absent: absence of H. pylori, present: presence of H. pylori.

).

3.5. Distribution of H. pylori with Respect to Gender and Age Group

H. pylori was detected in 55% of cases, with females accounting for 56.9% and males for 43.1% of the total positive cases. While females appeared to represent a higher proportion of H. pylori-positive cases. This difference was not statistically significant, suggesting no meaningful association between gender and H. pylori infection risk in this cohort.

Among 334 H. pylori-positive samples, the 31–40-year-old group accounted for the largest proportion of H. pylori-positive cases (n = 87, 26%). The 21–30-year-old group followed closely, contributing 78 (23.4%) positive cases (

Table 2. Gender and age-wise distribution of H. pylori.

Description	H. pylori n (%)
	Absent	Present
Gender
Male	124 (46.2)	144 (43.1)
Female	144 (53.8)	190 (56.9)
Age group
11–20	27 (10)	30 (9)
21–30	68 (25.3)	78 (23.3)
31–40	65 (24.3)	87 (26)
41–50	46 (17.2)	60 (18)
51–60	37 (13.8)	38 (11.4)
61–70	15 (5.6)	24 (7.2)
71–80	9 (3.4)	12 (3.6)
81–90	1 (0.4)	4 (1.2)
91–100	0	1 (0.3)
Total	268 (44.52)	334 (55.48)

).

3.6. Distribution of H. pylori with Respect to Inflammatory Response and Neutrophilic Activity

Histopathological evaluation of the 602 gastric biopsy samples demonstrated that the majority of cases exhibited mild chronic inflammation, accounting for 383 (63.6%). Moderate inflammation was noted in 207 (34.4%) of the samples, while severe inflammatory changes were relatively rare, observed in only 12 (2.0%) of cases. A statistically significant association was observed between H. pylori positivity and the degree of chronic inflammation in gastric biopsies (p < 0.00001). Among the H. pylori-positive cases (n = 334), moderate inflammation was the most frequent finding, present in 200 (59.9%) cases, followed by mild inflammation in 123 (36.8%) cases and severe inflammation in 11 cases (3.3%). Nearly all cases with moderate to severe inflammation were H. pylori-positive (

Table 3. Distribution of H. pylori vs. inflammation.

H. pylori n (%)	Inflammation n (%)
	Mild	Moderate	Severe	p-Value
Not detected	260 (97.0)	7 (2.6)	1 (0.4)	<0.00001
Detected	123 (36.8)	200 (59.9)	11 (3.3)

).

Assessment of neutrophilic activity, indicative of active gastritis, showed that mild activity was present in 150 (24.9%) of biopsies. Moderate and severe activities were less frequently observed, seen in 39 (6.5%) and 5 (0.8%) of cases, respectively. Notably, neutrophilic activity was absent in a substantial proportion of samples, 408 (67.8%). Neutrophilic activity also showed a highly significant correlation with H. pylori infection (p < 0.00001). Among the infected cases, 137 (41.0%) showed mild activity, 36 (10.8%) had moderate activity, and 5 (1.5%) showed severe activity. Notably, activity was absent in nearly half of the H. pylori-positive cases (46.7%, n = 156), indicating a spectrum of histologic response, ranging from chronic inactive to active gastritis. On the other hand, absence of activity was much more frequent in H. pylori-negative samples (252), reinforcing the link between bacterial presence and mucosal immune response (

Table 4. Distribution of H. pylori vs. neutrophilic activity.

H. pylori n (%)	Neutrophil Activity n (%)				p-Value
	Absent	Present
		Mild	Moderate	Severe
Not detected	252 (94.03)	13 (4.85)	3 (1.12)	0 (0)	<0.00001
Detected	156 (46.7)	137 (41.0)	36 (10.8)	5 (1.5)

).

3.7. Distribution of H. pylori with Respect to Intestinal Metaplasia and Gastric Atrophy

Intestinal metaplasia, a potential premalignant alteration, was identified in 7 (1.2%) of cases, whereas gastric atrophy, another marker of advanced gastric mucosal damage, was found in 4 (0.7%). Intestinal metaplasia and gastric atrophy were infrequent across all samples and did not show a statistically significant association with H. pylori status (p = 0.149 and p = 0.430, respectively). Intestinal metaplasia was present in 0.6% (95% CI; 0.02, p = 0.149) H. pylori-positive cases and 2% (95% CI; 5.37, p = 0.149) negative cases, while atrophy was noted in 0.9% (95% CI; 0.05, p = 0.430) positive cases and 0.4% (95% CI; 94.71, p = 0.430) negative cases (

Table 5. Intestinal metaplasia and gastric atrophy vs. H. pylori.

Histopathology vs. H. pylori	H. pylori	Absent n (%)	Present n (%)	95% Cl	OR	p-Value
Intestinal metaplasia vs. H. pylori	Not Detected	263 (98)	5 (2)	5.37	0.3	0.149
	Detected	332 (99.4)	2 (0.6)	0.02
Gastric atrophy vs. H. pylori	Not Detected	267 (99.6)	1 (0.4)	94.71	2.26	0.430
	Detected	331 (99.1)	3 (0.9)	0.05

).

4. Discussion

The observed prevalence of H. pylori infection (55%) aligns with previously reported Pakistani data (58–65%). This consistency validates the diagnostic accuracy achieved through combined histopathological and molecular methods. A study conducted by Abbasi et al. [16] in Islamabad reported a prevalence of 59.7% among patients undergoing endoscopy for chronic dyspeptic symptoms. Similarly, one research documented the prevalence of 56.6% [17]. At a broader level, a meta-analysis of South Asian countries revealed a pooled prevalence of 56.5% [18]. The prevalence in Pakistan has been documented to exceed 58% in the general population, with higher prevalence among individuals with lower socioeconomic status and poor sanitation [19].

In this study, 84.23% of samples were taken from the antrum, 2.3% from the body and 13.4% from both the antrum and body, with a prominent 55.13% of H. pylori-positive cases identified in antral biopsies. This anatomical site provides a relatively less acidic microenvironment and harbors a higher density of mucus-secreting cells, which facilitates bacterial adherence and survival [20]. Colonization of the antrum is the initiating event in the cascade of chronic gastritis [21]. One study reported that 74.6% of patients were biopsied from the antrum alone, highlighting the clinical emphasis on this region for H. pylori detection [22]. The updated Sydney classification system also recommends obtaining at least two biopsies from the antrum for optimal H. pylori detection.

Among H. pylori-positive biopsies, 59.9% exhibited moderate chronic inflammation, 10.8% demonstrated active neutrophilic infiltration, and there was a significant association with the presence of H. pylori (p < 0.00001). It has demonstrated a significant association between H. pylori positivity and both chronic inflammation and neutrophilic activity, with 55.6% of infected patients showing moderate to marked chronic inflammation, while 47% had histological activity [23], closely aligning with our observation.

A recent study observed that more than 50% of H. pylori-positive biopsies showed moderate to severe inflammation and active neutrophilic infiltration, emphasizing the inflammatory potential of H. pylori [24]. These findings are further supported by molecular data suggesting that the inflammatory response in H. pylori-associated gastritis is driven by activation of cytokine pathways and recruitment of immune cells, contributing to mucosal damage and disease progression [25]. Despite the high prevalence of mild inflammation and mild neutrophilic activity in H. pylori-positive cases, the frequency of intestinal metaplasia in our study was 0.6% (95% CI, 0.02, p = 0.149), and the frequency of gastric atrophy was 0.9% (95% CI, 0.05, p = 0.430) in H. pylori-positive cases. The low rates of intestinal metaplasia and atrophy align with studies from other developing regions where H. pylori is highly prevalent and many patients seek medical attention early because of symptoms like indigestion, which allows for earlier biopsy and diagnosis before severe tissue changes develop [26]. Furthermore, since the prevalence of intestinal metaplasia depends on the H. pylori-triggered neutrophilic activity and inflammation [27,28], most of the cases in the current study were in the category of mild inflammation and mild neutrophilic activity. There were a few cases among the category of moderate and severe, which are usually the pathological factors that accelerate the occurrence of intestinal metaplasia.

According to the correa cascade, the progression of H. pylori-induced chronic inflammation to atrophy, intestinal metaplasia, dysplasia, and eventually gastric cancer is a multistep, time-dependent process. The early detection of inflammation and activity without associated metaplasia or atrophy in our cohort reinforces the concept of a critical intervention window before irreversible changes occur. These findings agree with international data suggesting that metaplasia develops after long-term infection [8,29]. Moreover, the absence of significant metaplastic or atrophic changes may also reflect host and bacterial factors that influence disease progression, including host immune response, strain virulence, and environmental modifiers such as diet and smoking [29].

The majority of H. pylori-positive cases in this study were identified among individuals aged 11–40 years, highlighting a pattern of early life acquisition. These findings align with epidemiological trends reported globally, where infection is often acquired during childhood and persists into adulthood unless treated [30]. The observed decline in H. pylori-positive cases in older age groups (>50 years) may reflect a cohort effect where older individuals might have received treatment in the past, leading to bacterial eradication. Secondly, spontaneous bacterial clearance has been documented in some studies, particularly with aging-related changes in gastric physiology that create an inhospitable environment for H. pylori [31].

The slightly higher infection rate among females may result from sociocultural factors such as increased exposure during food preparation and caregiving roles. However, this difference was not statistically significant. These findings suggest no clear gender-based predisposition to H. pylori infection. Similar observations have been made in multiple population-based studies where gender did not emerge as a significant risk factor for H. pylori infection [32].

5. Conclusions

H. pylori infection is significantly associated with both chronic inflammation and active neutrophilic infiltration of the gastric mucosa, particularly in the antral region. Intestinal metaplasia and atrophy were uncommon in this study; however, their early identification remains crucial. Histopathological examination plays a central role in assessing the severity and potential progression of H. pylori-related gastric pathology.