Comparative Evaluation of Modified Giemsa and Warthin-Starry Stains for Helicobacter pylori Detection and Density Grading Using the Updated Sydney System

Abstract

Objective: Helicobacter pylori is one of the main causes of chronic gastritis, gastric ulcers, and stomach cancer. Accurate diagnosis of H. pylori infection is essential for effective treatment and prevention.

Methods: This research is an analytical observational study with a cross-sectional retrospective design that aims to compare histochemical special stains, Modified Giemsa, and Warthin-Starry for H. pylori Detection and Density Grading using the Updated Sydney System in 150 gastric biopsies of Chronic Gastritis at the Department of Anatomical Pathology, Dr. Wahidin Sudirohusodo General Hospital, Makassar, Indonesia.

Result: There was a significant difference in detection of H. pylori (p=0.001), Warthin-Starry detected more H. pylori positive 111 samples (74.00%) than Modified Giemsa positive 95 samples (63.30%) and there was a significant difference in Density Grading using the Updated Sydney System (p=0.001), Warthin-Starry staining showed a higher density score of Grade 2 (moderate) with 63 samples (42.00%), compared to Modified Giemsa, the most dominant density score of Grade 1 (mild) with 61 samples (40.70%).

Conclusion: Warthin-Starry detects more H. pylori compared to Modified Giemsa. Additionally, the Warthin-Starry yields higher Density Grading using the Updated Sydney System for Grade 2 (Moderate) compared to the Modified Giemsa, with a dominant grade at Grade 1 (Mild) in chronic gastritis. This highlights the importance of selecting the most appropriate histochemical staining method for accurate early diagnosis, where resource limitations restrict the use of advanced diagnostic modalities that may not be routinely available.

Introduction

Gastritis is a disease characterized by inflammation of the stomach mucosa. The etiological components are the Helicobacter pylori (H. pylori) agent. In cases of chronic gastritis, changes to the mucosa are most commonly associated with H. pylori-induced gastritis. The discovery of H. pylori demonstrated that gastric ulcers and chronic gastritis are caused by this bacterium [1]. H. pylori is a gram-negative, spiral-shaped bacterium, and a major etiological agent of chronic gastritis, peptic ulcers, and stomach cancer [2]. Accurate diagnosis of H. pylori infection is essential for effective treatment and prevention strategies [3], and this organism is also associated with stomach carcinoma and malignant lymphoma [4].

Although H. pylori can be visualized using hematoxylin & eosin (H&E) staining, its detection sensitivity and specificity are enhanced by special staining methods [5] such as Modified Giemsa, Warthin-Starry, and Immunohistochemistry (IHC) [6, 7], staining using H&E provides a basic visualization of gastric histology; however, due to limited contrast, it may fail to detect H. pylori in mild or patchy infections [8]. Special stains such as Modified Giemsa, Genta, Warthin-Starry silver, and IHC are more specific and sensitive [9, 10], and Modified Giemsa is a Romanowsky stain containing eosin, methylene blue II, and methylene azure II. The staining works by exploiting the chemical interaction between basic dyes and acidic cellular components, such as RNA & polysaccharides in the bacterial cell wall, resulting in a purplish-blue appearance of the bacteria. This staining method is simple, widely used, and cost-effective [9, 10]. The Warthin-Starry staining technique is a silver impregnation method that uses silver nitrate and formic acid. Silver interacts with parts of the bacterial cell wall, particularly peptidoglycan, causing silver metal to accumulate and making the bacteria look bigger and easier to see when viewed under a microscope, usually appearing black against a yellow or light brown background [9, 10]. The Updated Sydney System is generally used to classify gastritis based on histopathology. This system combines morphological, etiological, and topographical aspects. The assessment is semi-quantitative, covering atrophy, inflammation, H. pylori density, and intestinal metaplasia [11]. This is widely used worldwide, particularly in developing countries; however, it is not commonly used for routine examinations, including in Indonesia. In Indonesia, specifically at Dr. Wahidin Sudirohusodo Hospital in Makassar, one of the largest hospitals in Eastern Indonesia, the Anatomic Pathology Laboratory plays a crucial role in diagnosing gastritis. In 2016, a study at the hospital analyzed 162 stomach tissue samples; only 10 of them, or about 6.2% showed the presence of

H. pylori [12]. This low occurrence might be because of difficulties in diagnosis, such as depending on hematoxylin & eosin (H&E) staining alone and the lack of routine use of more sensitive techniques like Modified Giemsa or IHC. Research on the detection of H. pylori infection with Warthin-Starry compared to Modified-Giemsa of paraffin blocks of gastric biopsies in 2017 has been done [10], while comparative research on special histochemical staining of Modified Giemsa and Warthin-Starry for

H. pylori Detection and Density Grading using the Updated Sydney System in chronic gastritis in Makassar City, South Sulawesi Province, Indonesia has never been done. This research highlights the importance of selecting the most appropriate histochemical staining method for accurate early diagnosis, in clinical decision-making related to effective treatment of chronic gastritis [13], to more severe conditions, including strategies for preventing stomach carcinoma [14, 15], especially in resource-limited areas where advanced diagnostic techniques may not be routinely available. Therefore, accuracy for H. pylori Detection and Density Grading using the Updated Sydney System is crucial.

Aim of the Study

A comparative study of Modified Giemsa and Warthin-Starry stains for H. pylori detection and density grading using the Updated Sydney System in chronic gastritis.

Materials and Methods

1. Study design

The methodology is an analytical observational study with a cross-sectional retrospective design.

2. Population and Sample

The research population was 150 samples diagnosed by anatomic pathologists as chronic gastritis with positive and negative H. pylori results based on Hematoxylin Eosin and Giemsa staining, obtained from paraffin blocks of gastric biopsy tissues sent to the Anatomical Pathology Laboratory of Dr. Wahidin Sudirohosodo Hospital, Makassar, Indonesia, between January 2020 to December 2024.

Data Collection Techniques: 1). Collect and categorize all eligible samples based on histopathological examination diagnosed by anatomic pathologists as chronic gastritis with H. pylori positive and negative using Hematoxylin Eosin staining and Giemsa staining taken from angulus, antrum, antrum and corpus, antrum and angulus, antrum, angulus and corpus, antrum and cardia, and corpus sites from biopsies sent by clinicians. 2). Collect all eligible samples for paraffin blocks in order by registration number, then perform special histochemical staining examination to compare the evaluation of Modified Giemsa and Warthin-Starry for H. pylori detection and density grading using the Updated Sydney System.

3. Procedures

This research uses a special histochemically modified Giemsa stain; the detection of H. pylori will appear as bacterial colonies stained blue in chronic gastritis tissue, which is declared positive or negative through observation of the preparation using a light microscope and oil immersion. Objective criteria: (Nominal data scale) - Positive: there are blue-stained bacterial colonies in chronic gastritis tissue; - Negative: There are no blue- stained bacterial colonies in chronic gastritis tissue.

The detection of H. pylori using a special histochemical Warthin-Starry stain will appear as bacterial colonies stained black on a brown background and yellow to dark yellow in chronic gastritis tissue, which is declared as positive or negative through observation of the preparation using a light microscope with oil immersion. Objective criteria: (Nominal data scale) - Positive: there are bacterial colonies stained black with brown and yellow to dark yellow background in chronic gastritis tissue; - Negative: no bacterial colonies stained black with brown and yellow to dark yellow background in chronic gastritis tissue.

The Updated Sydney System is a combination of endoscopic and histologic findings. The density grading using the Updated Sydney System detection was compared between Modified Giemsa and Warthin-Starry in chronic gastritis expressed as descriptive: Grade 0 for None/ Normal, Grade 1 for Mild, Grade 2 for Moderate, and Grade 3 for Severe/Marked, through observation of preparations using a light microscope using oil immersion. Objective criteria: (Ordinal data scale) - Grade 0 for None/Normal: No H. pylori present anywhere; - Grade 1 for Mild: Only a few H. pylori in single or multiple foci; - Grade 2 for Moderate: Many H. pylori are seen in separate foci; - Grade 3 for Severe/Marked : >50% of the surface area is covered with H. pylori.

4. Statistical analyses

All data obtained from the research results were recorded, and then statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 29.0 (IBM Corp., Armonk, NY, USA). Categorical variables were described as numbers and percentages, and continuous variables were described as means with standard deviations or medians. 1). Univariate analysis, used to describe the characteristics of the data obtained, namely in the form of frequency distribution, ranges, and mean values presented in tabular form.

2). Bivariate analysis, consisting of: a). The Wilcoxon test is a non-parametric statistical test used to compare two groups of the same nominal data. In this study, the detection of H. pylori was expressed as positive and negative between the two groups of Modified Giemsa and Warthin-Starry. b). The Wilcoxon test is a non-parametric statistical test used to compare two groups of equal ordinal data. In this study, the density grading using the Updated Sydney System was assessed in the assessment of H. pylori density grading between two groups of Modified Giemsa and Warthin-Starry. All tests were two-tailed, and p-values <0.05 were considered statistically significant.

Results

1. General Characteristics of the Sample

The total number of samples was 150 Table 1 based on age category, with 84 samples (56.00%) in the >50 years age category and 66 samples (44.00%) in the <50 years age category. Based on gender, there were 84 male samples (56.0%) and 66 female samples (44.0%).

Table 1. General Characteristics of the Sample (n=150).

	Univariate analysis
Characteristics	Sample Quantity (n=150)	Percentage (%)
Age
> 50 Years	84	56
< 50 Years	66	44
Gender
Male	84	56
Female	66	44
Location
Angulus	1	0.7
Antrum	11	7.3
Antrum, corpus	30	20
Antrum, angulus	61	40.7
Antrum, angulus, corpus	41	27.3
Antrum, cardia	2	1.3
Corpus	4	2.7

Source: Primary Data, 2024

Meanwhile, based on the biopsy locations sent by clinicians, there were from the angulus 1 samples (0.70%), antrum 11 samples (7.30%), antrum and corpus 30 samples (20.00%), antrum and angulus 61 samples (40.70%), antrum, angulus, and corpus 41 samples (27.30%), antrum and cardia 2 samples (1.30%), and corpus 4 samples (2.70%).

2. General Characteristics of the H. pylori Detection

The results Table 2 of the H. pylori detection using Modified Giemsa was positive for 95 samples (63.30%) and negative for 55 samples (36.70%) while Warthin- Starry was positive for 111 samples (74.00%) and negative for 39 samples (26.00%) in chronic gastritis with a description of Histopathology results can be seen in Figure 1.

Table 2. General Characteristics and Comparison of Modified Giemsa and Warthin-Starry stains for Detection of H. pylori (n=150) .

Detection H. pylori			Univariate analysis			Bivariate analysis
	Modified-Giemsa			Warthin Starry		p-value
	Sample Quantity (n=150)	Percentage (%)		Sampel Quantity (n=150)	Percentage (%)
Positive bacteria	95	63.3		111	74,00	*0.001
Negative bacteria	55	36.7		39	26,00

* Wilcoxon test, statistically significant if the p value is less than 0.05.

Figure 1. Comparison Modified Giemsa and Warthin-Starry Stains for Detection of H. pylori Magnification (100x). a) Modified Giemsa: Positive Bacteria, b) Warthin-Starry: Positive Bacteria, c) Modified Giemsa: Negative Bacteria, d) Warthin-Starry: Negative Bacteria. Source: Primary Data, 2024.

3. General Characteristics of the H. pylori Density Grading using the Updated Sydney System

The results Table 3 of H. pylori density grading using the Updated Sydney System criteria between Modified Giemsa obtained Grade 0 (None/Normal) group as many as 55 samples (36.70%), Grade 1 (Mild) as many as 61 samples (40.70%), Grade 2 (Moderate) as many as 25 samples (16.70%) and Grade 3 (Severe/Marked) as many as 9 samples (6.00%) while Warthin-Starry obtained Grade 0 (None/Normal) group as many as 39 sample (26.00%), Grade 1 (Mild) as many as 14 samples (9.30%), Grade 2 (Moderate) as many as 63 samples (42.00%) and Grade 3 (Severe/Marked) as many as 34 samples (22.70%) in chronic gastritis with a description of Histopathology results can be seen in Figure 2.

Table 3. General Characteristics and Comparison of Modified Giemsa and Warthin-Starry stains for H. pylori Density Grading using the Updated Sydney System (n=150) .

H. pylori Density Grading using the Updated Sydney System			Univariate analysis			Bivariate analysis
	Modified-Giemsa			Warthin Starry		p-value
	Sample Quantity (n=150)	Percentage (%)		Sample Quantity (n=150)	Percentage (%)
Grade 0 (None/Normal)	55	36.7		39	26	*0.001
Grade 1 (Mild)	61	40.7		14	9.3
Grade 2 (Moderate)	25	16.7		63	42
Grade 3 (Severe/Marked)	9	6		34	22.7

* Wilcoxon test, statistically significant if the p value is less than 0.05.

Figure 2. Comparison Modified Giemsa and Warthin-Starry stains for H. pylori density grading using the Updated Sydney System magnification (100x). a) Modified Giemsa: Grade 0 (None/Normal),b) Warthin-Starry: Grade 0 (None/Normal), c) Modified Giemsa: Grade 1 (Mild), d) Warthin-Starry: Grade 1 (Mild), e) Modified Giemsa: Grade 2 (Moderate),f) Warthin-Starry: Grade 2 (Moderate), g) Modified Giemsa: Grade 3 (Severe/Marked), h) Warthin-Starry: Grade 3 (Severe/Marked). Source: Primary Data, 2024.

4. Comparison of the detection of H. pylori between Modified Giemsa and Warthin-Starry stains

Comparison of detection of H. pylori between Modified Giemsa and Warthin-Starry stains in chronic gastritis, the statistical results showed a significant mean difference of p=0.001 (p<0.05), which indicates that there is a significant difference. Where Warthin-Starry is more effective in detecting H. pylori, it obtained positive results in as many as 111 samples (74.00%) compared to Modified Giemsa, which obtained positive results in as many as 95 samples (63.30%) in chronic gastritis biopsies, with the results can be seen in Table 2.

5. Comparison of Modified Giemsa and Warthin-Starry stains for H. pylori density grading using Sydney Grading System

Comparison of Modified Giemsa and Warthin-Starry for H. pylori density grading using the Updated Sydney System in chronic gastritis, the statistical results showed a significant difference of p=0.001 (p<0.05), which indicates that there is a significant difference. Where Warthin-Starry produced a higher Density Score of the Updated Sydney System of Grade 2 (Moderate) as many as 63 samples (42.0%), compared to Modified Giemsa produced the most dominant Density Score of the Updated Sydney System of Grade 1 (Mild) as many as 61 samples (40.70%) in chronic gastritis biopsies with the results can be seen in Table 3.

Discussion

A total of 150 samples Table 1, the characteristics based on age category> 50 years were found the most, namely 84 samples (56.00%). These results are in line with the research of Breckan et al. (2016), who reported a population-based study involving all age groups that reported persistent H. pylori infection and suggested intrafamilial and environmental transmission routes. The results of this study support the implementation of community-level interventions [16], Everhart et al. (2000), noted that seroepidemiological studies in the US show ethnic disparities in the prevalence of H. pylori, with higher rates in certain minority groups, and social- environmental determinants likely contributing to the risk [3]. Meanwhile, in a study by Kamada et al. (2015), over 40 years in Japan, the prevalence of H. pylori and atrophic gastritis decreased with improvements in hygiene and living conditions, highlighting strong cohort and environmental effects [17]. In a study by Nakajima et al. (2010), over 17 years in Japan, the prevalence of H. pylori decreased alongside changes in gastrointestinal disease patterns, reflecting cohort effects and improvements in public health [18].

In addition, based on gender, 84 samples (56.00%) were male. These results are in line with the research of meta-analysis by Ibrahim et al. (2017), of 244 studies, which found differences in the prevalence of H. pylori according to gender in child and adult populations, suggesting biological and behavioral factors in the acquisition of infection [19]. A clinical study by Chuang et al. (2009) reinforced these findings chronic H. pylori infection is associated with differences in ghrelin and leptin levels according to gender. Hormonal changes may mediate the metabolic effects of long-term infection [20]. A study by Almashhadany, D.A., et al. (2023) reported a high prevalence of H. pylori with significant differences according to age and gender. The findings emphasize the need for context-appropriate screening and prevention strategies [21], and a study by Zamani et al. (2018) estimated that the prevalence of H. pylori is close to half of the world’s population, with significant regional heterogeneity, emphasizing the need for targeted prevention and control [22].

The results Table 2 of H. pylori detection between Warthin-Starry and Modified Giemsa showed that in detecting the presence of H. pylori, Warthin-Starry was positive as many as 111 samples (74.00%), while Modified Giemsa was positive as many as 95 samples (63.30%) in chronic gastritis biopsies, with Histopathology results picture can be seen in Figure 1. These results align with the study by Farouk et al. (2018), which reported that among histochemical stains, modified Giemsa balances sensitivity and ease of interpretation, while Warthin–Starry aids detection in biopsies with few bacteria but is more technically demanding [9], Krogfelt el al. (2005), the diagnostic testing for H. pylori emphasizes context-appropriate selection and combination of methods to improve accuracy; noninvasive tests require careful validation [23], Taha et al. (2018), where Immunohistochemistry shows higher sensitivity than conventional histochemical staining for H. pylori; the authors recommend immunohistochemistry as a supplement to diagnostically challenging biopsies [24], Pandya et al. (2013), the polymerase chain reaction test surpasses conventional staining in detecting H. pylori, identifying infections in several biopsies that are negative by conventional staining. This molecular test is applicable for confirmation and research [25]. Akeel et al. (2021) describe the evaluation of the diagnostic yield of immunohistochemistry (IHC) for H. pylori in patients in Saudi Arabia with minimal or atypical infection. IHC is superior to routine histochemical staining, especially in cases of low bacterial density, supporting its role as a confirmatory test [26]. Gowsik et al. (2019) explain that comparative research shows immunohistochemistry to be the most sensitive method for H. pylori; Giemsa remains a pragmatic option in resource-limited settings. The choice of method must balance accuracy and feasibility [6], the study by Kusters et al. (2006), elucidating bacterial virulence and host responses underlying gastritis, ulceration, and carcinogenesis, providing a mechanistic basis for targeted interventions [27], despite their importance and the study of Rokkas, T., et al. (2010), on his study showing that first-degree relatives of gastric cancer patients have a higher prevalence of H. pylori and poor gastric histology, targeted screening of high-risk families may be necessary [28].

The results Table 3 of H. pylori density grading using the Updated Sydney System using Warthin-Starry staining obtained Grade 2 (Moderate) in 63 samples (42.00%), while Modified Giemsa obtained Grade 1 (Mild) in 61 samples (40.70%). A description of the Histopathology results can be seen in Figure 2. These results are in line with research conducted by Sandhika et al. (2019), who concluded in the Indonesian cohort that Warthin–Starry and modified Giemsa stains are suitable for routine detection of H. pylori, although sensitivity varies; careful technique improves performance [10], Yadav et al. (2022), who found that Modified Giemsa is superior to some routine stains for screening, with IHK recognized as the benchmark for accuracy [5], Almeida et al. (2015), found in their study that certain H. pylori genotypes are associated with more severe gastric histopathology in Southern Europe. Genotyping can help classify the risk of mucosal damage [29]. The study by de Almeida et al. (2021) noted that variations in NOD2 in the host are associated with susceptibility to H. pylori infection, highlighting the role of innate immunity in disease risk. These findings support the integration of host genetics into epidemiological risk models [30], as a consideration of factors influencing the assessment of H. pylori density scores. Histological assessment was performed based on the latest Sydney system mentioned in the study by Miftahussurur et al. (2016), to rigorously validate indirect tests (serology, fecal antigen) in order to avoid biased prevalence estimates and recommend algorithms appropriate to the context [31]. These results align with research conducted by Misra et al. (2000), which states that the density and distribution of H. pylori vary topographically in the stomach and correlate with the degree of gastritis; multi-site sampling improves detection and assessment of the disease [32], and Jung et al. (2017), research, a meta-analysis in Korea showing that bismuth-based regimens and some non-bismuth quadruple regimens achieve better eradication rates than the old triple therapy regimen. Selection should reflect local resistance patterns [33], and Oling et al. (2015) study in a tertiary hospital with limited resources showed that dyspepsia patients have a high prevalence of H. pylori, confirming the need for easily accessible diagnostics and appropriate management protocols [34].

Comparison of Modified Giemsa and Warthin-Starry for H. pylori detection in chronic gastritis, the statistical results showed a significant mean difference of p=0.001 (p<0.05), which indicates that there is a significant difference in H. pylori detection between Modified Giemsa and Warthin-Starry stains in chronic gastritis. Where Warthin-Starry is more effective in detecting the presence of H. pylori, it obtained positive results in as many as 111 samples (74.00%) compared to Modified Giemsa, which obtained positive results in as many as 95 samples (63.30%) in chronic gastritis biopsies, with the results can be seen in Table 2 and Figure 1. These results are in line with research conducted by Mujtaba et al. (2025), which explains the comparison of current diagnostic techniques supporting the integrated use of invasive and non-invasive tests to maximize detection and guide effective therapy [35], Farouk et al. (2018), his study among histochemical stains, modified Giemsa balances sensitivity and ease of interpretation, while Warthin–Starry aids detection in biopsies with few bacteria but is more technically demanding [9], Krogfelt et al. (2005), the diagnostic testing for H. pylori emphasizes context-appropriate selection and combination of methods to improve accuracy; noninvasive tests require careful validation [23]. This is attributed to Warthin-Starry being an argentaffin (silver-based) staining method, which allows staining of H. pylori into a solid black color against a pale yellow background making it easier to detect, especially in cases with low bacterial density, whereas Modified Giemsa uses aniline-based dyes (azure B and eosin), which rely more on the color contrast of the cytoplasm and bacteria. As a result, it is easier to miss in cases of mild infection or at low densities. The advantages of Warthin-Starry detection over Modified Giemsa in chronic gastritis are associated with high sensitivity in detecting H. pylori, visualization ability in low-density cases, and high accuracy in histopathological confirmation, although this method requires specialized techniques and is more expensive. Modified Giemsa remains relevant for rapid and practical screening, but is less accurate as a single method in cases of mild infection or advanced chronicity.

Comparison of Modified Giemsa and Warthin-Starry stains for H. pylori density grading using the Updated Sydney System in chronic gastritis, statistical results showed a significant difference of p=0.001 (p<0.05). Where Warthin-Starry produced a higher density Score of the Updated Sydney System of Grade 2 (moderate) as many as 63 samples (42.00%) compared to Modified Giemsa produced the most dominant Density Score of the Updated Sydney System of Grade 1 (mild) as many as 61 samples (40.70%) in chronic gastritis biopsies with the results can be seen in Table 3 and Figure 2. These results are consistent with research conducted by Kocsmár, É., et al. (2017), in which Giemsa sensitivity to H. pylori decreased in low inflammatory activity, while IHC and FISH maintained higher detection rates. Additional testing is recommended when inflammation is minimal [36], as explained by Yakoob, J., et al. (2005), who noted that combining rapid urease testing with histopathology enhances the reliability of H. pylori diagnosis in developing countries, mitigating the limitations of using a single test alone [8]. In a study by Although in a study by Nurdin et al. (2016), IHC was superior to Giemsa in detecting H. pylori and correlated with morphological changes in active chronic gastritis [37], and Laheij et al (2000), demonstrated that without a single gold standard, latent class analysis showed that histology, culture, and rapid urease testing were accurate, with the best performance when combined. A multi-test strategy minimizes classification errors [38]. This was attributed to Warthin-Starry using silver staining and high contrast methods, which enabled the visualization of even small, weakly staining forms, or cocci, making it superior in detecting very low densities. In contrast, Modified Giemsa is effective for the detection of spiral forms of H. pylori at moderate to high densities, but fails to recognize cocci or very low-density forms that often appear in chronic infection or are on partial treatment.

Emphasis on the importance of choosing the most appropriate histochemical staining method for accurate early diagnosis in clinical decision-making. In addition to sensitivity, the selection of staining techniques for routine use must consider cost efficiency and technical feasibility, especially in resource-limited areas where advanced diagnostic techniques may not be readily available, particularly in regions such as Makassar, Indonesia. Sandhika et al. (2019); Taha et al. (2018), in their study, found that although Warthin-Starry showed higher sensitivity, this method is more expensive, more technically complex, and time-consuming compared to Modified Giemsa staining, which is simpler and more cost-effective. This makes Modified Giemsa a practical and adequate option for initial screening. However, in cases with high clinical suspicion of H. pylori infection but negative or unclear results on Modified Giemsa staining, or for accurate density assessment in research settings, Warthin-Starry is highly sensitive, although immunohistochemistry (IHC) is considered the gold standard for detection [10, 24]. Similarly, Kocsmár, É., et al. (2017) found in their study that Giemsa staining was less sensitive for detecting H. pylori than immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), and that many cases of H. pylori were not detected when using Giemsa alone [36].

The Warth-Starry stain highlights the crucial role of accurate histopathological diagnosis as the primary line of defense in identifying patients at high risk for H. pylori- associated sequelae, including stomach cancer. This aligns with the ongoing global effort to improve early cancer detection. More sensitive H. pylori detection is a crucial step in identifying individuals at high risk for stomach cancer, the goal that aligns with recent findings by Noto et al. (2012) and de Brito et al. (2018) on the molecular pathogenesis of H. pylori-induced carcinogenesis [39, 40]. The recent research by Wang et al. (2025) focusing on the discovery of novel molecular biomarkers [41], and the study by Díaz et al. (2024), Matsuoka et al. (2023), and Mirzaei et al. (2024) on the development of advanced biosensing platforms utilizing innovative biomaterials for ultra-sensitive detection [42, 43, 44]. However, these advanced technologies are often not routinely available in resource-limited areas such as Makassar City, South Sulawesi Province, Indonesia. Therefore, optimizing and validating affordable and readily available methods, such as the comparative evaluation of histochemical dyes presented in this research, remains a pragmatic and essential strategy to bridge this technological gap.

The accurate grading of H. pylori density not only guides immediate clinical management but also helps stratify patients for more intensive surveillance, potentially bridging the gap until more advanced molecular or point-of-care tests become accessible and affordable in these regions. Regarding effective treatment of chronic gastritis and more severe conditions, including strategies for preventing stomach carcinoma, Malfertheiner, P., et al. (2022) found in their study that Maastricht IV/ Florence 2012 confirmed the eradication of H. pylori as the primary strategy for preventing stomach ulcers and stomach carcinoma [14], the 2015 Kyoto Report by Sugano, K., et al. (2015) emphasized that H. pylori gastritis is a disease that requires treatment and introduced the Kyoto Classification, emphasizing that staging gastritis through the Updated Sydney System to assess the risk of stomach carcinoma, which is the basis for a global strategy for stomach carcinoma prevention through H. pylori eradication [15]. Rugge, M., et al. (2007). The OLGA staging system is a practical method for assessing the risk of stomach carcinoma based on the degree and location of gastritis atrophy. This system can provide clinical guidance for monitoring patients, especially after H. pylori eradication [13].

In conclusion, warthin-Starry detects more H. pylori compared to Modified Giemsa. Additionally, the Warthin- Starry yields higher Density Grading using the Updated Sydney System for Grade 2 (moderate) compared to the Modified Giemsa, with a dominant grade at Grade 1 (mild) in chronic gastritis. This highlights the importance of selecting the most appropriate histochemical staining method for accurate early diagnosis, where resource limitations restrict the use of advanced diagnostic modalities may not be routinely available.

Declarations Funding Statement

This research was personally funded by the author and supported by the Specialist-Subspecialist Doctor Education Scholarship Program, Ministry of Health of the Republic of Indonesia.

Clinical trial registration

Not applicable.

Conflict of Interest/Competing interests

The authors declare no conflicts of interest.

Code availability

Not applicable.

Authors’ contributions

R.F. contributed to data collection and primary drafting of the manuscript. U.A.M., M.H.C. contributed to the manuscript’s conception, study design, and final drafting, and supervised the research. U.A.M. and M.H.C. contributed to data interpretation and manuscript editing.

U.A.M. and M.H.C. contributed to histopathological evaluation and analysis. S.W. and J. supervised the research. S.T. contributed to statistical analysis and interpretation. A.Y. contributed to the Anatomical Pathology Laboratories at Dr. Wahidin Sudirohusodo Hospital. All authors reviewed and approved the final version of the manuscript.

Ethical Approval

The research has obtained ethical approval and was conducted in accordance with the Declaration of Helsinki. The confidentiality of the research data was guaranteed, and the research procedures and protocols were approved by the Health Research Ethics Commission of the Faculty of Medicine, Hasanuddin University, Makassar, Indonesia (Approval Number: 726/UN4.6.4.5.31-/PP36/2024).

Acknowledgments

The authors would like to thank all who supported this research.

• The author wishes to thank all the teachers in the Anatomical Pathology Specialist Program, Department of Anatomical Pathology, Faculty of Medicine Hasanuddin University, Makassar, Indonesia.

• The author also wants to express appreciation to the Department of the Anatomical Pathology at Dr. Wahidin Sudirohusodo Hospital in Makassar, Indonesia, for their help in collecting the samples.

If approved by any scientific body, it becomes part of the thesis approved by the student of Anatomical Pathology Specialist Program, Department of Anatomical Pathology, Faculty of Medicine Hasanuddin University, Makassar, Indonesia.

Declaration on generative AI and AI-assisted technologies in the writing process: The authors affirm that no generative AI or AI-assisted technologies were used in the preparation of this manuscript; all writing, analysis, and revisions were performed by the authors.