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.
<italic id="italic-33e9cc5f9d7e14ca64497280e923652a">Aim of the Study<italic id="italic-8"/></italic>
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.
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).