Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies

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However, comparative data on their diagnostic performance and cost-effectiveness in detecting Helicobacter pylori are limited. This study aimed to assess the feasibility of using H&E and AB/PAS as alternatives to Giemsa. Methods A retrospective study was conducted on 816 gastric biopsy cases collected between 2019 and 2021. Three slides (H&E, Giemsa, and AB/PAS) were previously prepared from each paraffin-embedded tissue sample and blindly evaluated by three independent examiners. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic accuracy were calculated using 2×2 contingency tables. Results H&E showed a sensitivity of 51.6%, specificity of 74.4%, and diagnostic accuracy of 66.4%, while AB/PAS had a sensitivity of 45.9%, specificity of 73.2%, and accuracy of 63.7%. H&E was the most cost-effective and fastest method; AB/PAS was the most expensive and time-consuming. Giemsa demonstrated superior diagnostic performance. Conclusions H&E offers practical benefits for screening but lacks sufficient sensitivity for definitive diagnosis. AB/PAS is less effective and less economical. Giemsa remains the most reliable stain for H. pylori detection. Combining H&E with Giemsa may optimize both efficiency and diagnostic accuracy. Further prospective studies are warranted. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-1148/v1", "name": "Assessing the diagnostic accuracy of routine hematoxylin and eosin,..." } } ] } Home Browse Assessing the diagnostic accuracy of routine hematoxylin and eosin,... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Alwahaibi N, Al Mamari AM, Al Aamri A et al. Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.12688/f1000research.170290.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] Nasar Alwahaibi https://orcid.org/0000-0002-9421-0951 1 , Al-Mutaz Al Mamari 1 , Arwa Al Aamri 1 , Yaqeen Al Sulimani 1 , Alwaleed Al Balushi 1 Nasar Alwahaibi https://orcid.org/0000-0002-9421-0951 1 , Al-Mutaz Al Mamari 1 , [...] Arwa Al Aamri 1 , Yaqeen Al Sulimani 1 , Alwaleed Al Balushi 1 PUBLISHED 21 Oct 2025 Author details Author details 1 Department of Biomedical Science, Sultan Qaboos University College of Medicine and Health Science, Muscat, 123, Oman Nasar Alwahaibi Roles: Conceptualization, Formal Analysis, Investigation, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Al-Mutaz Al Mamari Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Arwa Al Aamri Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Yaqeen Al Sulimani Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Alwaleed Al Balushi Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background Hematoxylin and Eosin (H&E), Alcian Blue/Periodic Acid-Schiff (AB/PAS), and Giemsa stains are routinely used in the histopathological evaluation of gastric biopsies. However, comparative data on their diagnostic performance and cost-effectiveness in detecting Helicobacter pylori are limited. This study aimed to assess the feasibility of using H&E and AB/PAS as alternatives to Giemsa. Methods A retrospective study was conducted on 816 gastric biopsy cases collected between 2019 and 2021. Three slides (H&E, Giemsa, and AB/PAS) were previously prepared from each paraffin-embedded tissue sample and blindly evaluated by three independent examiners. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic accuracy were calculated using 2×2 contingency tables. Results H&E showed a sensitivity of 51.6%, specificity of 74.4%, and diagnostic accuracy of 66.4%, while AB/PAS had a sensitivity of 45.9%, specificity of 73.2%, and accuracy of 63.7%. H&E was the most cost-effective and fastest method; AB/PAS was the most expensive and time-consuming. Giemsa demonstrated superior diagnostic performance. Conclusions H&E offers practical benefits for screening but lacks sufficient sensitivity for definitive diagnosis. AB/PAS is less effective and less economical. Giemsa remains the most reliable stain for H. pylori detection. Combining H&E with Giemsa may optimize both efficiency and diagnostic accuracy. Further prospective studies are warranted. READ ALL READ LESS Keywords Helicobacter pylori, gastric biopsy, Giemsa stain, hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, sensitivity, specificity Corresponding Author(s) Nasar Alwahaibi ( [email protected] ) Close Corresponding author: Nasar Alwahaibi Competing interests: No competing interests were disclosed. Grant information: Financial support to conduct the study was obtained from the Deanship of Research at Sultan Qaboos University in Oman, with the UF/MED/BIOM/24/01. The funder did not have any role in the study design, data collection, analysis, interpretation of data, or writing the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Alwahaibi N et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Alwahaibi N, Al Mamari AM, Al Aamri A et al. Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.12688/f1000research.170290.1 ) First published: 21 Oct 2025, 14 :1148 ( https://doi.org/10.12688/f1000research.170290.1 ) Latest published: 10 Jan 2026, 14 :1148 ( https://doi.org/10.12688/f1000research.170290.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction Helicobacter pylori ( H. pylori ) is a significant global health concern, with nearly half of the world’s population infected by this bacterium. 1 H. pylori is a gram-negative, spiral-shaped bacterium that colonizes the gastric mucosa and is a major cause of chronic gastritis, peptic ulcers, and gastric cancer. 2 It plays a pivotal role in the pathogenesis of various gastric disorders, making its accurate identification critical in clinical practice. Due to its high sensitivity and specificity, as well as additional histopathological information, histopathological demonstration is one of the most reliable methods for detecting H. pylori , particularly in gastric biopsy specimens. 3 Studying H. pylori is essential not only for improving diagnostic methods and understanding its pathogenic mechanisms but also for developing effective treatment strategies to mitigate its widespread impact on public health. Hematoxylin and Eosin (H&E), Alcian Blue/Periodic Acid-Schiff (AB/PAS), and Giemsa staining methods are essential routine stains for the histopathological evaluation of gastric biopsies. A survey was conducted into the use of H&E and special stains on gastric specimens in histopathology departments within the National Health Service, United Kingdom. One hundred and sixty-seven histopathology departments in the UK were contacted using an e-mail questionnaire. Gastric specimens are stained using H&E in 47% of departments and 53% use H&E combined with special stains (AB/PAS, and Giemsa). 4 These stains play a critical role in diagnosing various gastric conditions, including the identification of H. pylori. H&E provides an overall assessment of tissue architecture, AB/PAS highlights mucin alterations, and Giemsa specifically stains H. pylori , making them complementary tools in gastric pathology. Giemsa staining is widely regarded as a specific, cheap, easy to perform and reliable method for detecting H. pylori in gastric biopsies. Its ability to clearly highlight the bacterium against the background tissue makes it a preferred choice in gastric pathology. 5 – 8 However, its limitation lies in its specificity, as it is not suitable for general histopathological evaluation. H&E is the most commonly used routine stain in histopathology due to its ability to provide excellent contrast between cellular and extracellular structures, making it a valuable tool for general tissue examination. Certain laboratories can optimize H&E staining to enhance the detection of H. pylori in gastric biopsies. 9 , 10 However, its lack of specificity in differentiating between certain cellular components or microorganisms is a notable limitation. AB/PAS is primarily employed to differentiate between acidic and neutral mucins in the gastric epithelium. Periodic Acid-Schiff (PAS) reaction targets 1,2-glycol groups present in carbohydrates, oxidizing them to form aldehydes, which then react with Schiff’s reagent to produce a magenta color. 11 The outer membrane of H. pylori contains carbohydrate-rich structures, such as polysaccharides and glycans, that include 1,2-glycol groups. 12 , 13 However, the amount and accessibility of 1,2-glycol groups in H. pylori may vary among strains, this will affect the consistency of detection with PAS. If its utility in identifying H. pylori can be established, it might serve as an adjunct to specific staining methods like Giemsa, providing broader pathological insights. Although these staining techniques are widely used, there is a notable lack of comprehensive studies comparing their diagnostic efficacy, accuracy, staining time, and cost-effectiveness. This gap in the literature highlights the need for further investigation. This study seeks to address this by evaluating the strengths and limitations of H&E, AB/PAS, and Giemsa stains in gastric biopsies. Therefore, this study aimed to evaluate the feasibility of utilizing H&E and AB/PAS staining methods as potential alternatives to the Giemsa staining method for the detection of H. pylori in gastric biopsies. Materials and methods Study setting This retrospective cohort study was conducted in the Pathology Department, Sultan Qaboos University Hospital, utilizing gastric biopsy cases collected between 2019 and 2021. The data were accessed for research purposes on June 11, 2024. Demographic data for the selected cases were obtained from the Trak-care system. The slides were stored in appropriate boxes at room temperature, ensuring they were not exposed to sunlight or humidity to preserve their quality. Sample selection The sample selection was based on inclusion and exclusion criteria to ensure the reliability of the comparative analysis. To be included in the study, each case was required to have a complete set of slides stained with the three selected methods: H&E, Giemsa, and AB/PAS. Cases were excluded if they lacked any one of the required stained slides, were repeated submissions of the same patient (duplicates), or involved non-gastric biopsy specimens, as the study focused exclusively on gastric tissue. Out of the initial pool of 1,370 cases reviewed, 544 did not meet the inclusion criteria and were excluded. Therefore, a total of 816 cases with complete and eligible staining data were included in the final analysis. Cost and staining procedures As this was a retrospective study, three glass slides were previously prepared from each paraffin-embedded gastric tissue biopsy. Each slide was stained using one of the three standard histological methods: H&E, Giemsa, or AB/PAS. For H&E, sections were stained with Harris hematoxylin (300 mL per staining dish; Cellavision, Cat# 361075) for 8 minutes at room temperature, followed by eosin Y (300 mL per staining dish; Surgipath, Leica Biosystems, Cat# 3801619) for 4 minutes at room temperature. The total staining time was approximately 15–25 minutes. Reagent costs for both dyes were obtained from local suppliers. For Giemsa, sections were stained with Giemsa working solution prepared in pH 6.8 buffer (single-slide dish, 50 mL; Merck, Sigma-Aldrich, Cat# GS500) for 25 minutes at room temperature. The procedure required approximately 30–45 minutes. For AB/PAS, sections were sequentially stained with 1% Alcian blue, pH 2.5 (200 μL per slide; Fluka Analytical, Sigma-Aldrich, Cat# 33864-99-2) for 20 minutes at room temperature; periodic acid (200 μL per slide; Merck, Sigma-Aldrich, Cat# 375810-25 G) for 5 minutes at room temperature; and Schiff reagent (200 μL per slide; Carl Roth, Cat# X900.2) for 8 minutes at room temperature. The full staining process took approximately 40–60 minutes. Costs for Alcian blue, periodic acid, and Schiff’s reagent were obtained from local suppliers. All staining procedures were performed according to standard protocols under identical laboratory conditions. Evaluation An independent examiner evaluated each stain without knowledge of the sample to reduce bias. In cases of uncertainty, a thorough examination was performed using an oil immersion objective at 1,000 × magnification. Particularly difficult cases were further reviewed by an expert examiner. Each stained slide was microscopically examined (Nikon, Module Eclipse Ei R, Kanagawa, Japan) to identify the presence of H. pylori. Statistical analysis All statistical analyses were performed using IBM SPSS Statistics 25. (IBM Corp. Released 2023. IBM SPSS Statistics for Windows, Version 25. Armonk, NY: IBM Corp). To evaluate the diagnostic performance of the H&E and AB/PAS staining methods in detecting H. pylori , we constructed 2 × 2 contingency tables using the Giemsa stain as the reference standard. From these tables, we calculated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic accuracy for each method. In addition, Receiver Operating Characteristic (ROC) curve analysis was conducted to assess and compare the diagnostic performance of the staining techniques. The Area Under the Curve (AUC) was calculated for each method, providing a comprehensive measure that incorporates both sensitivity and specificity across all possible thresholds. AUC values were interpreted as follows: excellent (≥0.90), good (0.80–0.89), fair (0.70–0.79), and poor (<0.70). Differences in AUC values between stains were evaluated using DeLong’s test, with p-values < 0.05 considered statistically significant. Ethical considerations Ethical approval for this study was obtained from the Medical Research Ethics Committee (MREC) at Sultan Qaboos University, Muscat, Oman, under study number 3040. The committee is responsible for overseeing research ethics and ensuring compliance with institutional and national standards. All procedures involving human participants, materials, or data were conducted in accordance with national regulations and the principles of the Declaration of Helsinki. As the study involved a retrospective analysis of archived staining slides and associated demographic data, the requirement for informed consent was waived by the ethics committee. During data collection, the authors had access to potentially identifying information through the Trak-care system; however, all data were anonymized before analysis, and no identifiable information is included in the final dataset or publication. Participant confidentiality was strictly maintained throughout the study. Results A total of 816 gastric biopsy cases were included in the study. The gender distribution was nearly equal, with 417 (51.1%) males and 399 (48.9%) females. The majority of patients were between 41–60 years of age (36.9%), followed by those aged 61–80 years (29.2%). Younger age groups, including 1–20 years and 21–40 years, represented 9.8% and 21.7% of the cases, respectively, while only 2.5% were in the 81–88 age range. Chronic gastritis was the most frequently observed condition, present in 590 cases (72.3%). Intestinal metaplasia was identified in 162 cases (19.9%), while lymphoplasmacytic infiltrate was found in 304 cases (37.3%). Hyperplastic changes were observed in 102 cases (12.5%). Gastric adenocarcinoma was diagnosed in 25 cases, accounting for 3.1% of the total, and other pathological findings such as inflammatory, regenerative changes, preneoplastic lesions, and neoplastic/tumour-like lesions, were reported in 212 cases (26%) ( Table 1 ). Table 1. Demographic and histopathological characteristics of the studied gastric biopsy cases. Demographic characteristics and differential diagnosis Subcategory Total (N = 816) Gender Male 417 (51.1%) Female 399 (48.9%) Age in Years 1– 20 80 (9.8%) 21– 40 177 (21.7%) 41– 60 301 (36.9%) 61– 80 238 (29.2%) 81– 88 20 (2.5%) Chronic gastritis Presence 590 (72.3%) Absence 226 (27.7%) Intestinal metaplasia Presence 162 (19.9%) Absence 654 (80.1%) Lymphoplasmacytic infiltrate Presence 304 (37.3%) Absence 512 (62.7%) Hyperplasia Presence 102 (12.5%) Absence 714 (87.5%) Gastric adenocarcinoma presence 25 (3.1%) Absence 791 (96.9%) Others Presence 212 (26%) Absence 604 (74%) When compared to Giemsa staining, AB/PAS identified 131 of the Giemsa-positive cases as positive, while 142 Giemsa-negative cases were also marked as positive by AB/PAS. Conversely, 154 Giemsa-positive cases were missed (false negatives), and 389 Giemsa-negative cases were correctly identified as negative. For H&E staining, 147 of the Giemsa-positive cases were correctly identified, with 136 false positives. H&E missed 138 Giemsa-positive cases, and 395 Giemsa-negative cases were confirmed as negative. These results indicate that H&E had slightly higher agreement with Giemsa staining than AB/PAS, showing better sensitivity and a slightly higher overall diagnostic concordance in detecting H. pylori ( Table 2 ). Table 2. Comparison of Hematoxylin & Eosin and Alcian Blue/Periodic Acid-Schiff’s Staining Methods with Giemsa for Detection of H. pylori. Stain method Giemsa Positive Giemsa Negative Total AB/PAS Positive 131 142 273 AB/PAS Negative 154 389 543 H&E Positive 147 136 283 H&E Negative 138 395 533 The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy (DA) of H&E and AB/PAS staining methods were compared against Giemsa staining as the reference standard for detecting H. pylori in gastric biopsies. H&E staining demonstrated a sensitivity of 51.6%, specificity of 74.4%, PPV of 51.9%, NPV of 74.1%, and an overall diagnostic accuracy of 66.4%. In contrast, AB/PAS showed a slightly lower performance, with a sensitivity of 45.9%, specificity of 73.2%, PPV of 47.9%, NPV of 71.6%, and diagnostic accuracy of 63.7% ( Table 3 ). Table 3. Comparison of Hematoxylin & Eosin and Alcian Blue/Periodic Acid-Schiff’s diagnostic performance against Giemsa for H. pylori detection. Parameters H&E AB/PAS Sensitivity 51.6% 45.9% Specificity 74.4% 73.2% Positive Predictive Value 51.9% 47.9% Negative Predictive Value 74.1% 71.6% Diagnostic Accuracy 66.4% 63.7% The diagnostic performance of H&E and AB/PAS staining methods for detecting H. pylori was further evaluated using Receiver Operating Characteristic (ROC) curve analysis, with Giemsa staining as the reference standard. The Area Under the Curve (AUC) for H&E was 0.63, indicating modest discriminatory ability. In comparison, the AB/PAS stain showed a slightly lower AUC of 0.60, reflecting limited diagnostic performance. Both curves were positioned only slightly above the no-discrimination line (AUC = 0.50), suggesting that while H&E performs marginally better than AB/PAS, neither stain provides high diagnostic accuracy on its own [ Figure 1 ]. Figure 2 shows representative images of H. pylori identified in gastric tissue sections stained with H&E, AB/PAS, and Giemsa. Figure 1. Receiver operating characteristic curve analysis for comparing the diagnostic accuracy of Hematoxylin and Eosin, Alcian Blue/Periodic Acid-Schiff, staining methods for the detection of H. pylori , using Giemsa staining as reference. Figure 2. Representative images showing the presence of H. pylori in stained gastric tissue sections using Hematoxylin and Eosin (A), Alcian Blue/Periodic Acid-Schiff (B), and Giemsa stain (C) at 40x magnification. When demonstrating H. pylori in paraffin-embedded gastric tissue sections, H&E is the most cost-effective method, with an estimated reagent cost of USD 0.10–0.20 per slide. This is due to its routine use and reliance on inexpensive, ready-to-use reagents. Giemsa stain has a moderately higher cost, ranging from USD 0.10–0.25 per slide. In contrast, the AB/PAS method is the most expensive, with a per-slide cost of USD 0.45–0.95, primarily due to the use of multiple high-cost reagents such as Alcian Blue and Schiff’s reagent. In terms of time efficiency, H&E also has the shortest staining time, requiring approximately 15 – 25 minutes. Giemsa takes moderately longer, around 30 – 45 minutes, due to preparation and staining steps. AB/PAS is the most time-consuming, taking approximately 40 – 60 minutes because of its multi-step protocol and multiple reagent incubations ( Table 4 ). Table 4. Comparison cost and staining time for Hematoxylin & Eosin, Giemsa, and Alcian Blue/Periodic Acid-Schiff’s stains. Stains Reagent/Stain Dilution used and bottle size Cost in OMR and USD Estimated cost per slide in OMR and USD Estimated staining time in minutes Comments H & E Harris H Ready to use, 1000 ml 31.9 82.75 0.038-0.077 0.10 – 0.20 15 – 25 Lowest cost and fastest method. Eosin Y Ready to use, 500 ml 14.8 38.40 Giemsa Stain Giemsa 50%, 500 ml 38.9 101 0.038 – 0.096 0.10 – 0.25 30 – 45 Moderate cost and staining time. AB/PAS Alcian Blue 8GX 1%, 25 g 171 444 0.17 – 0.37 0.45 – 0.95 40 – 60 Highest cost and longest staining time. Periodic Acid 1%, 25 g 51.2 133 Schiff’s Reagent Ready to use, 1 L 22.1 57.4 Discussion The primary aim of this study was to assess the diagnostic performance, staining efficiency, and cost-effectiveness of H&E and AB/PAS staining methods in comparison to the Giemsa stain, which is commonly regarded as the reference standard for detecting H. pylori in gastric biopsy specimens. This multifaceted evaluation was intended to guide laboratories in selecting appropriate staining protocols that balance diagnostic reliability with operational efficiency, particularly in settings with limited resources or high specimen volumes. The findings highlight that while H&E staining is cost-effective and time-efficient, its diagnostic performance for H. pylori is limited. H&E staining demonstrated a relatively low sensitivity of 51.6%, indicating that it correctly identified just over half of the true positive H. pylori cases confirmed by Giemsa staining. Its specificity was moderate at 74.4%, reflecting a fair ability to correctly identify negative cases. The positive predictive value of H&E was 51.9%, suggesting that only slightly more than half of the positive results detected by H&E were true positives. Conversely, its negative predictive value was 74.1%, implying a moderate level of confidence in ruling out H. pylori when H&E results are negative. The overall diagnostic accuracy of H&E compared to Giemsa was 66.4%, reflecting moderate agreement between the two stains. These findings suggest that H&E staining is more reliable for excluding H. pylori rather than confirming its presence and should not be used as a standalone diagnostic tool. Combining H&E with Giemsa may improve detection accuracy in routine practice. Our results are consistent with other studies, such as one from Qassim University, Saudi Arabia, which reported higher sensitivity (66.7%) and specificity (91.2%) though our values were lower. 14 Similar findings were also reported. 5 In fact, the reported sensitivity of identifying H. pylori in gastric specimens using only H&E-stained slides ranges from 66% when evaluated by general histopathologists to as high as 90% when assessed by expert pathologists. 15 , 16 In addition, H. pylori was detected in 37% (14 out of formalin fixed, paraffin wax embedded tissue from 38 gastric biopsy) stained with H&E staining method. 7 A study evaluating 325 gastric biopsies from 65 patients with preneoplastic lesions found that H&E had a positivity rate of 41.5% to 49% for detecting H. pylori , which was lower than Giemsa staining across all anatomical sites (61.5%–72%). 17 Another retrospective study in 390 gastric biopsies showed that H&E staining method has 67% sensitivity in mild gastritis and 83% in moderate or severe gastritis. 18 Our findings are in disagreement with other study where they reported that the diagnostic accuracy of H&E was 91.7%, with a sensitivity of 93.2%, and a specificity of 86.7%. However, the gold standard chosen for their study was culture of antral tissue biopsies rather than Giemsa stain. 19 A study comparing five staining methods, haematoxylin and eosin (H&E), immunohistochemistry (IHC), silver staining (HpSS), Alcian yellow-toluidine blue (Leung) method (A–Y), and Genta stain, for detecting H. pylori in 118 gastric biopsies found no significant difference in the efficacy of H&E, IHC, HpSS, and A–Y. The authors concluded that H&E is sufficient for the initial evaluation of gastric biopsies in patients with upper gastrointestinal symptoms, as it is a well-established, cost-effective, and simple method that offers quick processing and highly reproducible results. 20 Another prospective study found that H&E staining is highly effective for identifying H. pylori in gastric biopsies. Among 613 biopsies, 71.1% were clearly negative for H. pylori on H&E and did not require further staining. Of the inconclusive H&E cases, only a small proportion (15.9%) were toluidine blue positive. In addition, most H&E-positive cases for H. pylori were confirmed by toluidine blue staining. The authors concluded that routine use of special stains is unnecessary, as H&E evaluation with selective use of additional stains is sufficient to detect nearly all cases of H. pylori gastritis. 21 Despite this variability, several studies support the role of H&E as a cost-effective and rapid screening tool. Some research indicates that H&E can achieve higher diagnostic accuracy when assessed by experienced pathologists or when used selectively in combination with other stains like Giemsa or toluidine blue. In particular, studies have shown that special stains may be unnecessary in clear negative or positive cases identified on H&E, supporting a more targeted staining approach in clinical practice. Overall, the collective evidence suggests that while H&E should not be used as a standalone diagnostic tool for H. pylori , it remains a valuable first-line method when combined with selective confirmatory staining to enhance diagnostic accuracy and cost-efficiency. In this study, AB/PAS staining method demonstrated the lowest overall diagnostic performance for H. pylori detection among the evaluated techniques. It had the longest staining time (40–60 minutes) and the highest reagent cost (approximately USD 0.45 – 0.95 per slide), largely due to multiple incubation steps involving high-cost reagents. Diagnostic metrics further reflected its limitations: AB/PAS showed a sensitivity of 45.9%, indicating it missed more than half of true positive cases compared to the Giemsa gold standard. The specificity was moderate at 73.2%, reflecting a fair ability to correctly identify negative cases. However, the positive predictive value was relatively low at 47.9%, suggesting a high chance of false positives, while the negative predictive value was 71.6%, showing moderate reliability in ruling out infections. Its overall diagnostic accuracy was only 63.7%, likely influenced by the stain’s tendency to highlight mucins and background components that can obscure the bacteria or mimic its appearance, leading to diagnostic ambiguity. Supporting evidence from the literature reinforces these findings. For instance, a study, which included forty-five formalin-fixed, paraffin-embedded blocks of gastric biopsies, reported higher sensitivity (81.8%) and specificity (86.9%) for AB/PAS, though still lower than more targeted stains such as Giemsa (reference) and Gimenez stains (88.9% sensitivity and 100% specificity). 22 In contrast, another study reported even lower sensitivity (40%) and specificity (67.65%) for AB/PAS, highlighting its inconsistent performance across different studies. 14 These discrepancies underline the influence of technique variability and interpretive challenges when using AB/PAS. Overall, despite its routine availability and utility in highlighting mucins and intestinal metaplasia, AB/PAS staining lacks the diagnostic precision needed for reliable detection of H. pylori. Its low sensitivity and modest accuracy suggest it should not be used as a standalone method. Instead, it may serve a complementary role alongside more accurate stains like Giemsa, especially in cases requiring concurrent evaluation of gastric mucosal changes. The Giemsa stain is probably one of the most popular stains because of its simplicity and good contrast. 5 , 23 – 25 This study presents several key strengths. It provides a direct comparison of three routinely used histological stains, H&E, Giemsa, and AB/PAS, for detecting H. pylori , addressing a notable gap in the existing literature. The inclusion of a large sample size (816 gastric biopsy cases) enhances the statistical validity and generalizability of the results. Furthermore, the study evaluates practical parameters such as staining time and reagent cost, offering important insights into the cost-effectiveness and feasibility of each method in routine histopathology practice. This study has several limitations. First, the retrospective nature of the study, which relied on histological presentations collected between 2019 and 2021, introduces inherent biases. These include inconsistent data collection, potential slide degradation over time, and variations in staining protocols or processing techniques. Such factors may compromise the reproducibility and reliability of microscopic evaluations. Second, the investigation was confined to a single institution, which is the Sultan Qaboos University Hospital, over a fixed three-year period. This narrow scope raises concerns about selection bias and limits the applicability of the findings to broader clinical settings. Differences in diagnostic protocols, patient demographics, and laboratory practices in other institutions may influence the detection and interpretation of H. pylori. Third, while Giemsa staining was used as the reference (gold standard) method, relying on a single stain for validation may introduce classification bias. Giemsa itself may be subject to misinterpretation, especially in cases with low bacterial density, staining artifacts, or faded slides. This may have influenced the perceived diagnostic accuracy of the H&E and AB/PAS methods. Lastly, despite measures taken to reduce observer bias, such as assigning each staining method to a different pathologist and consulting a senior expert for unclear cases, the potential for inter-observer variability in interpreting H. pylori presence remains a notable limitation. The results of this study offer meaningful implications across multiple levels. They inform diagnostic decision-making by clarifying the performance limitations of common staining techniques, guiding pathologists on when additional confirmatory testing is warranted. In clinical practice, the findings support more efficient use of existing resources by identifying cost-effective screening options. For institutions, these results can support evidence-based revisions to laboratory protocols and staff training programs aimed at enhancing diagnostic accuracy for H. pylori. Furthermore, the study reinforces the importance of standardized staining procedures to reduce diagnostic variability and improve patient outcomes. Conclusions While H&E proved to be the most cost-effective and time-efficient stain, its limited sensitivity highlights its inadequacy as a standalone diagnostic method for H. pylori. AB/PAS demonstrated the lowest diagnostic accuracy, high cost, and longer staining time, making it less practical for targeted H. pylori detection. Giemsa remains the most reliable stain due to its superior sensitivity and specificity. The findings underscore the importance of using Giemsa either alone or in combination with H&E for improved diagnostic confidence. Laboratories aiming to balance efficiency with diagnostic accuracy should consider H&E for screening and reserve Giemsa for confirmatory purposes. Further multi-institutional and prospective research is recommended to validate these findings and guide best practices for H. pylori detection in routine histopathology. Reporting guidelines Zenodo: STROBE Statement for Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies. https://doi.org/10.5281/zenodo.17051552 27 Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Data availability Underlying data Zenodo: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies. https://doi.org/10.5281/zenodo.17330903 26 This project contains the following underlying data: • Data set for evaluation of H. pylori using hematoxylin and eosin, Alcian blue/periodic acid–Schiff, and Giemsa staining in 816 gastric biopsy samples. • Supplementary figure 1. Representative hematoxylin and eosin (H&E)–stained gastric tissue sections (40×) showing the presence and absence of spiral-shaped Helicobacter pylori : (A) negative for H. pylori ; (B) scanty organisms; (C) abundant organisms; (D) highly abundant organisms. • Supplementary figure 2. Representative Alcian blue/periodic acid–Schiff (AB/PAS)–stained gastric tissue sections (40×) showing the presence and absence of spiral-shaped Helicobacter pylori : (A) negative for H. pylori ; (B) scanty organisms; (C) abundant organisms; (D) highly abundant organisms. • Supplementary figure 3. Representative Giemsa-stained gastric tissue sections (40×) showing the presence and absence of spiral-shaped Helicobacter pylori : (A) negative for H. pylori ; (B) scanty organisms; (C) abundant organisms; (D) highly abundant organisms. Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). References 1. World Gastroenterology Organization: Global guideline: Helicobacter pylori in developing countries. J. Dig. Dis. 2011; 12 : 319–326. Publisher Full Text 2. Warren JR, Marshall B: Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983; 321 : 1273–1275. Publisher Full Text 3. 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Wright CL, Kelly JK: The use of routine special stains for upper gastrointestinal biopsies. Am. J. Surg. Pathol. 2006; 30 : 357–361. Publisher Full Text 22. Yadav R, Sagar M: Comparison of different histological staining methods for detection of Helicobacter pylori infection in gastric biopsy. Cureus. 2022; 14 : e27316. PubMed Abstract | Publisher Full Text | Free Full Text 23. Cha MS: Comparative analysis of histochemical stains about detection of H. pylori in gastric mucosa. Korean J. Clin. Lab. Sci. 2007; 39 : 223–230. 24. El-Zimaity HM, Segura AM, Genta RM, et al. : Histologic assessment of Helicobacter pylori status after therapy: comparison of Giemsa, Diff-Quik, and Genta stains. Mod. Pathol. 1998; 11 : 288–291. PubMed Abstract 25. Moayyedi P, Dixon MF: Any role left for invasive tests? Histology in clinical practice. Gut. 1998; 43 (Suppl 1): S51–S55. PubMed Abstract | Publisher Full Text | Free Full Text 26. Alwahaibi N, Al Mamari A-M, Al Aamri A, et al. : Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies. Zenodo. 2025. Publisher Full Text 27. Alwahaibi N, Al Mamari A, Al Aamri A, et al. : Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies. Zenodo. 2025. Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 21 Oct 2025 ADD YOUR COMMENT Comment Author details Author details 1 Department of Biomedical Science, Sultan Qaboos University College of Medicine and Health Science, Muscat, 123, Oman Nasar Alwahaibi Roles: Conceptualization, Formal Analysis, Investigation, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Al-Mutaz Al Mamari Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Arwa Al Aamri Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Yaqeen Al Sulimani Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Alwaleed Al Balushi Roles: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation Competing interests No competing interests were disclosed. Grant information Financial support to conduct the study was obtained from the Deanship of Research at Sultan Qaboos University in Oman, with the UF/MED/BIOM/24/01. The funder did not have any role in the study design, data collection, analysis, interpretation of data, or writing the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 10 Jan 2026, 14:1148 https://doi.org/10.12688/f1000research.170290.2 version 1 Published: 21 Oct 2025, 14:1148 https://doi.org/10.12688/f1000research.170290.1 Copyright © 2025 Alwahaibi N et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Alwahaibi N, Al Mamari AM, Al Aamri A et al. Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.12688/f1000research.170290.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 21 Oct 2025 Views 0 Cite How to cite this report: Liu H. Reviewer Report For: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.5256/f1000research.187732.r435909 ) The direct URL for this report is: https://f1000research.com/articles/14-1148/v1#referee-response-435909 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 30 Dec 2025 Hengrui Liu , Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Informatization, Guangzhou, China Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.187732.r435909 In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really ... Continue reading READ ALL In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really dial back the implication that this approach already meaningfully inform diagnosis or patient outcome, and instead frame it more clearly as a comparative methodological assessment with downstream relevance rather than immediate application. Shortening some claims would help. Just a little restraint here go a long way. The background section need more grounding in disease biology and clinical context. Helicobacter pylori is not just a technical detection problem, it is a causal driver of gastric carcinogenesis and disease progression, and that connection should be stated clearly and early so the reader understand why this question matter beyond the lab bench. A brief discussion of how H. pylori infection contribute to gastric cancer development and behavior would strengthen the motivation a lot, and it would be very natural to weave in evidence showing its role in migration and clinical outcome, such as what is described in “Helicobacter pylori infection facilitates cell migration and potentially impact clinical outcomes in gastric cancer”. Right now the background feel slightly procedural, and I think anchoring it more firmly in cancer biology would improve flow and relevance. In the methods, things are mostly clear, though some choices feel under-explained. I find myself wondering why certain thresholds and comparisons are treated as self-evident when they are not, especially for readers outside pathology. A bit more justification, not more detail but more reasoning, would help. The writing here is fine but feel slightly rushed, and tightening the logic rather than adding length would make it stronger. The results section present a lot of numbers and comparisons, and while the data appear consistent, the narrative interpretation drift a bit. At times it sound like performance differences are more decisive than they really are. I would recommend slowing down the interpretation and reminding the reader what question is actually being answered at each step. Some repetition is okay here, but clarity matter more. Right now it feel like the data talk faster than the conclusions can keep up. In the discussion, this is where most of the overreach happen. The study do something useful, but it do not yet redefine practice. I would strongly encourage the authors to separate what the data directly show from what they hope might be true in future settings. There is a tendency to generalize beyond the cohort and beyond the technical scope, and pulling that back would actually make the paper more credible. A bit more self-critique, especially around generalizability and clinical translation, would be welcome. It almost get there, but not quite. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Liu H. Reviewer Report For: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.5256/f1000research.187732.r435909 ) The direct URL for this report is: https://f1000research.com/articles/14-1148/v1#referee-response-435909 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Jan 2026 Nasar Alwahaibi , Department of Biomedical Science, Sultan Qaboos University College of Medicine and Health Science, Muscat, 123, Oman 10 Jan 2026 Author Response We would like to take this opportunity to express our thanks to the reviewer for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of ... Continue reading We would like to take this opportunity to express our thanks to the reviewer for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of the reviewer. Reviewer comments In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really dial back the implication that this approach already meaningfully inform diagnosis or patient outcome, and instead frame it more clearly as a comparative methodological assessment with downstream relevance rather than immediate application. Shortening some claims would help. Just a little restraint here go a long way. Response As suggested, we revised the Conclusions section of the abstract to use more cautious language. The findings are now presented as informing possible approaches and future research, rather than making immediate diagnostic. We also clarified that the study represents a methodological assessment and that further validation is needed before clinical use. The background section need more grounding in disease biology and clinical context. Helicobacter pylori is not just a technical detection problem, it is a causal driver of gastric carcinogenesis and disease progression, and that connection should be stated clearly and early so the reader understand why this question matter beyond the lab bench. A brief discussion of how H. pylori infection contribute to gastric cancer development and behavior would strengthen the motivation a lot, and it would be very natural to weave in evidence showing its role in migration and clinical outcome, such as what is described in “Helicobacter pylori infection facilitates cell migration and potentially impact clinical outcomes in gastric cancer”. Right now the background feel slightly procedural, and I think anchoring it more firmly in cancer biology would improve flow and relevance. Response As suggested, we have revised the Introduction section to clearly state H. pylori's causal link to gastric cancer development and its implications for disease progression and clinical outcomes. We have incorporated a brief discussion on how H. pylori infection contributes to gastric cancer, including a reference to the type of evidence highlighted in your comment (e.g., its role in cell migration and clinical outcomes). In the methods, things are mostly clear, though some choices feel under-explained. I find myself wondering why certain thresholds and comparisons are treated as self-evident when they are not, especially for readers outside pathology. A bit more justification, not more detail but more reasoning, would help. The writing here is fine but feel slightly rushed, and tightening the logic rather than adding length would make it stronger. Response We appreciate your comment regarding the Methods section. We agree that providing more justification and reasoning for certain choices enhances clarity, especially for non-specialist readers. As suggested, we have added brief explanations for why Giemsa was selected as the reference standard and provided context for the chosen AUC interpretation thresholds. We also reinforced the rationale behind blinding and expert review to enhance the logical flow and address any perception of the writing being rushed. The results section present a lot of numbers and comparisons, and while the data appear consistent, the narrative interpretation drift a bit. At times it sound like performance differences are more decisive than they really are. I would recommend slowing down the interpretation and reminding the reader what question is actually being answered at each step. Some repetition is okay here, but clarity matter more. Right now it feel like the data talk faster than the conclusions can keep up. Response As suggested, we have carefully re-examined the Results section. We have focused on slowing down the interpretation, explicitly linking each finding back to the study's core questions (e.g., assessing H&E and AB/PAS as alternatives to Giemsa), and using more tempered language when discussing performance differences. In the discussion, this is where most of the overreach happen. The study do something useful, but it do not yet redefine practice. I would strongly encourage the authors to separate what the data directly show from what they hope might be true in future settings. There is a tendency to generalize beyond the cohort and beyond the technical scope, and pulling that back would actually make the paper more credible. A bit more self-critique, especially around generalizability and clinical translation, would be welcome. It almost get there, but not quite. Response As suggested, we have thoroughly revised the Discussion to address these points. We have focused on strengthening the self-critique regarding generalizability and clinical translation, ensuring that the interpretations are grounded explicitly in the data presented. Thank you. We would like to take this opportunity to express our thanks to the reviewer for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of the reviewer. Reviewer comments In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really dial back the implication that this approach already meaningfully inform diagnosis or patient outcome, and instead frame it more clearly as a comparative methodological assessment with downstream relevance rather than immediate application. Shortening some claims would help. Just a little restraint here go a long way. Response As suggested, we revised the Conclusions section of the abstract to use more cautious language. The findings are now presented as informing possible approaches and future research, rather than making immediate diagnostic. We also clarified that the study represents a methodological assessment and that further validation is needed before clinical use. The background section need more grounding in disease biology and clinical context. Helicobacter pylori is not just a technical detection problem, it is a causal driver of gastric carcinogenesis and disease progression, and that connection should be stated clearly and early so the reader understand why this question matter beyond the lab bench. A brief discussion of how H. pylori infection contribute to gastric cancer development and behavior would strengthen the motivation a lot, and it would be very natural to weave in evidence showing its role in migration and clinical outcome, such as what is described in “Helicobacter pylori infection facilitates cell migration and potentially impact clinical outcomes in gastric cancer”. Right now the background feel slightly procedural, and I think anchoring it more firmly in cancer biology would improve flow and relevance. Response As suggested, we have revised the Introduction section to clearly state H. pylori's causal link to gastric cancer development and its implications for disease progression and clinical outcomes. We have incorporated a brief discussion on how H. pylori infection contributes to gastric cancer, including a reference to the type of evidence highlighted in your comment (e.g., its role in cell migration and clinical outcomes). In the methods, things are mostly clear, though some choices feel under-explained. I find myself wondering why certain thresholds and comparisons are treated as self-evident when they are not, especially for readers outside pathology. A bit more justification, not more detail but more reasoning, would help. The writing here is fine but feel slightly rushed, and tightening the logic rather than adding length would make it stronger. Response We appreciate your comment regarding the Methods section. We agree that providing more justification and reasoning for certain choices enhances clarity, especially for non-specialist readers. As suggested, we have added brief explanations for why Giemsa was selected as the reference standard and provided context for the chosen AUC interpretation thresholds. We also reinforced the rationale behind blinding and expert review to enhance the logical flow and address any perception of the writing being rushed. The results section present a lot of numbers and comparisons, and while the data appear consistent, the narrative interpretation drift a bit. At times it sound like performance differences are more decisive than they really are. I would recommend slowing down the interpretation and reminding the reader what question is actually being answered at each step. Some repetition is okay here, but clarity matter more. Right now it feel like the data talk faster than the conclusions can keep up. Response As suggested, we have carefully re-examined the Results section. We have focused on slowing down the interpretation, explicitly linking each finding back to the study's core questions (e.g., assessing H&E and AB/PAS as alternatives to Giemsa), and using more tempered language when discussing performance differences. In the discussion, this is where most of the overreach happen. The study do something useful, but it do not yet redefine practice. I would strongly encourage the authors to separate what the data directly show from what they hope might be true in future settings. There is a tendency to generalize beyond the cohort and beyond the technical scope, and pulling that back would actually make the paper more credible. A bit more self-critique, especially around generalizability and clinical translation, would be welcome. It almost get there, but not quite. Response As suggested, we have thoroughly revised the Discussion to address these points. We have focused on strengthening the self-critique regarding generalizability and clinical translation, ensuring that the interpretations are grounded explicitly in the data presented. Thank you. Competing Interests: The authors declare no competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Jan 2026 Nasar Alwahaibi , Department of Biomedical Science, Sultan Qaboos University College of Medicine and Health Science, Muscat, 123, Oman 10 Jan 2026 Author Response We would like to take this opportunity to express our thanks to the reviewer for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of ... Continue reading We would like to take this opportunity to express our thanks to the reviewer for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of the reviewer. Reviewer comments In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really dial back the implication that this approach already meaningfully inform diagnosis or patient outcome, and instead frame it more clearly as a comparative methodological assessment with downstream relevance rather than immediate application. Shortening some claims would help. Just a little restraint here go a long way. Response As suggested, we revised the Conclusions section of the abstract to use more cautious language. The findings are now presented as informing possible approaches and future research, rather than making immediate diagnostic. We also clarified that the study represents a methodological assessment and that further validation is needed before clinical use. The background section need more grounding in disease biology and clinical context. Helicobacter pylori is not just a technical detection problem, it is a causal driver of gastric carcinogenesis and disease progression, and that connection should be stated clearly and early so the reader understand why this question matter beyond the lab bench. A brief discussion of how H. pylori infection contribute to gastric cancer development and behavior would strengthen the motivation a lot, and it would be very natural to weave in evidence showing its role in migration and clinical outcome, such as what is described in “Helicobacter pylori infection facilitates cell migration and potentially impact clinical outcomes in gastric cancer”. Right now the background feel slightly procedural, and I think anchoring it more firmly in cancer biology would improve flow and relevance. Response As suggested, we have revised the Introduction section to clearly state H. pylori's causal link to gastric cancer development and its implications for disease progression and clinical outcomes. We have incorporated a brief discussion on how H. pylori infection contributes to gastric cancer, including a reference to the type of evidence highlighted in your comment (e.g., its role in cell migration and clinical outcomes). In the methods, things are mostly clear, though some choices feel under-explained. I find myself wondering why certain thresholds and comparisons are treated as self-evident when they are not, especially for readers outside pathology. A bit more justification, not more detail but more reasoning, would help. The writing here is fine but feel slightly rushed, and tightening the logic rather than adding length would make it stronger. Response We appreciate your comment regarding the Methods section. We agree that providing more justification and reasoning for certain choices enhances clarity, especially for non-specialist readers. As suggested, we have added brief explanations for why Giemsa was selected as the reference standard and provided context for the chosen AUC interpretation thresholds. We also reinforced the rationale behind blinding and expert review to enhance the logical flow and address any perception of the writing being rushed. The results section present a lot of numbers and comparisons, and while the data appear consistent, the narrative interpretation drift a bit. At times it sound like performance differences are more decisive than they really are. I would recommend slowing down the interpretation and reminding the reader what question is actually being answered at each step. Some repetition is okay here, but clarity matter more. Right now it feel like the data talk faster than the conclusions can keep up. Response As suggested, we have carefully re-examined the Results section. We have focused on slowing down the interpretation, explicitly linking each finding back to the study's core questions (e.g., assessing H&E and AB/PAS as alternatives to Giemsa), and using more tempered language when discussing performance differences. In the discussion, this is where most of the overreach happen. The study do something useful, but it do not yet redefine practice. I would strongly encourage the authors to separate what the data directly show from what they hope might be true in future settings. There is a tendency to generalize beyond the cohort and beyond the technical scope, and pulling that back would actually make the paper more credible. A bit more self-critique, especially around generalizability and clinical translation, would be welcome. It almost get there, but not quite. Response As suggested, we have thoroughly revised the Discussion to address these points. We have focused on strengthening the self-critique regarding generalizability and clinical translation, ensuring that the interpretations are grounded explicitly in the data presented. Thank you. We would like to take this opportunity to express our thanks to the reviewer for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of the reviewer. Reviewer comments In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really dial back the implication that this approach already meaningfully inform diagnosis or patient outcome, and instead frame it more clearly as a comparative methodological assessment with downstream relevance rather than immediate application. Shortening some claims would help. Just a little restraint here go a long way. Response As suggested, we revised the Conclusions section of the abstract to use more cautious language. The findings are now presented as informing possible approaches and future research, rather than making immediate diagnostic. We also clarified that the study represents a methodological assessment and that further validation is needed before clinical use. The background section need more grounding in disease biology and clinical context. Helicobacter pylori is not just a technical detection problem, it is a causal driver of gastric carcinogenesis and disease progression, and that connection should be stated clearly and early so the reader understand why this question matter beyond the lab bench. A brief discussion of how H. pylori infection contribute to gastric cancer development and behavior would strengthen the motivation a lot, and it would be very natural to weave in evidence showing its role in migration and clinical outcome, such as what is described in “Helicobacter pylori infection facilitates cell migration and potentially impact clinical outcomes in gastric cancer”. Right now the background feel slightly procedural, and I think anchoring it more firmly in cancer biology would improve flow and relevance. Response As suggested, we have revised the Introduction section to clearly state H. pylori's causal link to gastric cancer development and its implications for disease progression and clinical outcomes. We have incorporated a brief discussion on how H. pylori infection contributes to gastric cancer, including a reference to the type of evidence highlighted in your comment (e.g., its role in cell migration and clinical outcomes). In the methods, things are mostly clear, though some choices feel under-explained. I find myself wondering why certain thresholds and comparisons are treated as self-evident when they are not, especially for readers outside pathology. A bit more justification, not more detail but more reasoning, would help. The writing here is fine but feel slightly rushed, and tightening the logic rather than adding length would make it stronger. Response We appreciate your comment regarding the Methods section. We agree that providing more justification and reasoning for certain choices enhances clarity, especially for non-specialist readers. As suggested, we have added brief explanations for why Giemsa was selected as the reference standard and provided context for the chosen AUC interpretation thresholds. We also reinforced the rationale behind blinding and expert review to enhance the logical flow and address any perception of the writing being rushed. The results section present a lot of numbers and comparisons, and while the data appear consistent, the narrative interpretation drift a bit. At times it sound like performance differences are more decisive than they really are. I would recommend slowing down the interpretation and reminding the reader what question is actually being answered at each step. Some repetition is okay here, but clarity matter more. Right now it feel like the data talk faster than the conclusions can keep up. Response As suggested, we have carefully re-examined the Results section. We have focused on slowing down the interpretation, explicitly linking each finding back to the study's core questions (e.g., assessing H&E and AB/PAS as alternatives to Giemsa), and using more tempered language when discussing performance differences. In the discussion, this is where most of the overreach happen. The study do something useful, but it do not yet redefine practice. I would strongly encourage the authors to separate what the data directly show from what they hope might be true in future settings. There is a tendency to generalize beyond the cohort and beyond the technical scope, and pulling that back would actually make the paper more credible. A bit more self-critique, especially around generalizability and clinical translation, would be welcome. It almost get there, but not quite. Response As suggested, we have thoroughly revised the Discussion to address these points. We have focused on strengthening the self-critique regarding generalizability and clinical translation, ensuring that the interpretations are grounded explicitly in the data presented. Thank you. Competing Interests: The authors declare no competing interests. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Gimiga N. Reviewer Report For: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.5256/f1000research.187732.r429654 ) The direct URL for this report is: https://f1000research.com/articles/14-1148/v1#referee-response-429654 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 25 Nov 2025 Nicoleta Gimiga , Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Approved VIEWS 0 https://doi.org/10.5256/f1000research.187732.r429654 The manuscript presents a large retrospective analysis comparing three widely used histological stains—H&E, Giemsa, and AB/PAS—for the detection of Helicobacter pylori in gastric biopsies. The topic is relevant, as histological diagnosis remains a cornerstone in H. pylori detection, and ... Continue reading READ ALL The manuscript presents a large retrospective analysis comparing three widely used histological stains—H&E, Giemsa, and AB/PAS—for the detection of Helicobacter pylori in gastric biopsies. The topic is relevant, as histological diagnosis remains a cornerstone in H. pylori detection, and the study addresses a practical gap concerning diagnostic performance and cost-effectiveness. Strenghts: - Large sample size The inclusion of 816 biopsy cases is a major strength and provides statistical reliability and generalizability. Direct comparison of three staining methods -Few studies offer such a direct and comprehensive comparison. -Addresses a genuine gap in routine diagnostic pathology. Blind assessment- The use of three independent examiners helps minimize observer bias. LIMITATIONS: Retrospective design Use of Giemsa as the sole “gold standard” The manuscript is promising and clinically relevant , with strong methodological foundations and valuable practical implications. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pediatrics, Pediatric gastroenterologyst I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gimiga N. Reviewer Report For: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.5256/f1000research.187732.r429654 ) The direct URL for this report is: https://f1000research.com/articles/14-1148/v1#referee-response-429654 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 21 Oct 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 2 (revision) 10 Jan 26 read Version 1 21 Oct 25 read read Nicoleta Gimiga , Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Hengrui Liu , Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Informatization, Guangzhou, China Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Liu H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 Jan 2026 | for Version 2 Hengrui Liu , Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Informatization, Guangzhou, China 0 Views copyright © 2026 Liu H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have clearly addressed the major issues raised in the earlier round, and the manuscript now read much more smoothly, and the manuscript is now suitable for indexing. Competing Interests No competing interests were disclosed. Reviewer Expertise cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Liu H. Peer Review Report For: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.5256/f1000research.194814.r449128) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-1148/v2#referee-response-449128 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Liu H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 30 Dec 2025 | for Version 1 Hengrui Liu , Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Informatization, Guangzhou, China 0 Views copyright © 2026 Liu H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really dial back the implication that this approach already meaningfully inform diagnosis or patient outcome, and instead frame it more clearly as a comparative methodological assessment with downstream relevance rather than immediate application. Shortening some claims would help. Just a little restraint here go a long way. The background section need more grounding in disease biology and clinical context. Helicobacter pylori is not just a technical detection problem, it is a causal driver of gastric carcinogenesis and disease progression, and that connection should be stated clearly and early so the reader understand why this question matter beyond the lab bench. A brief discussion of how H. pylori infection contribute to gastric cancer development and behavior would strengthen the motivation a lot, and it would be very natural to weave in evidence showing its role in migration and clinical outcome, such as what is described in “Helicobacter pylori infection facilitates cell migration and potentially impact clinical outcomes in gastric cancer”. Right now the background feel slightly procedural, and I think anchoring it more firmly in cancer biology would improve flow and relevance. In the methods, things are mostly clear, though some choices feel under-explained. I find myself wondering why certain thresholds and comparisons are treated as self-evident when they are not, especially for readers outside pathology. A bit more justification, not more detail but more reasoning, would help. The writing here is fine but feel slightly rushed, and tightening the logic rather than adding length would make it stronger. The results section present a lot of numbers and comparisons, and while the data appear consistent, the narrative interpretation drift a bit. At times it sound like performance differences are more decisive than they really are. I would recommend slowing down the interpretation and reminding the reader what question is actually being answered at each step. Some repetition is okay here, but clarity matter more. Right now it feel like the data talk faster than the conclusions can keep up. In the discussion, this is where most of the overreach happen. The study do something useful, but it do not yet redefine practice. I would strongly encourage the authors to separate what the data directly show from what they hope might be true in future settings. There is a tendency to generalize beyond the cohort and beyond the technical scope, and pulling that back would actually make the paper more credible. A bit more self-critique, especially around generalizability and clinical translation, would be welcome. It almost get there, but not quite. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 10 Jan 2026 Nasar Alwahaibi, Department of Biomedical Science, Sultan Qaboos University College of Medicine and Health Science, Muscat, 123, Oman We would like to take this opportunity to express our thanks to the reviewer for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of the reviewer. Reviewer comments In the abstract, the overall structure work fine but the confidence level feel a bit too high given what actually get tested. Right now it read close to a clinical conclusion, and that make me uneasy. I would really dial back the implication that this approach already meaningfully inform diagnosis or patient outcome, and instead frame it more clearly as a comparative methodological assessment with downstream relevance rather than immediate application. Shortening some claims would help. Just a little restraint here go a long way. Response As suggested, we revised the Conclusions section of the abstract to use more cautious language. The findings are now presented as informing possible approaches and future research, rather than making immediate diagnostic. We also clarified that the study represents a methodological assessment and that further validation is needed before clinical use. The background section need more grounding in disease biology and clinical context. Helicobacter pylori is not just a technical detection problem, it is a causal driver of gastric carcinogenesis and disease progression, and that connection should be stated clearly and early so the reader understand why this question matter beyond the lab bench. A brief discussion of how H. pylori infection contribute to gastric cancer development and behavior would strengthen the motivation a lot, and it would be very natural to weave in evidence showing its role in migration and clinical outcome, such as what is described in “Helicobacter pylori infection facilitates cell migration and potentially impact clinical outcomes in gastric cancer”. Right now the background feel slightly procedural, and I think anchoring it more firmly in cancer biology would improve flow and relevance. Response As suggested, we have revised the Introduction section to clearly state H. pylori's causal link to gastric cancer development and its implications for disease progression and clinical outcomes. We have incorporated a brief discussion on how H. pylori infection contributes to gastric cancer, including a reference to the type of evidence highlighted in your comment (e.g., its role in cell migration and clinical outcomes). In the methods, things are mostly clear, though some choices feel under-explained. I find myself wondering why certain thresholds and comparisons are treated as self-evident when they are not, especially for readers outside pathology. A bit more justification, not more detail but more reasoning, would help. The writing here is fine but feel slightly rushed, and tightening the logic rather than adding length would make it stronger. Response We appreciate your comment regarding the Methods section. We agree that providing more justification and reasoning for certain choices enhances clarity, especially for non-specialist readers. As suggested, we have added brief explanations for why Giemsa was selected as the reference standard and provided context for the chosen AUC interpretation thresholds. We also reinforced the rationale behind blinding and expert review to enhance the logical flow and address any perception of the writing being rushed. The results section present a lot of numbers and comparisons, and while the data appear consistent, the narrative interpretation drift a bit. At times it sound like performance differences are more decisive than they really are. I would recommend slowing down the interpretation and reminding the reader what question is actually being answered at each step. Some repetition is okay here, but clarity matter more. Right now it feel like the data talk faster than the conclusions can keep up. Response As suggested, we have carefully re-examined the Results section. We have focused on slowing down the interpretation, explicitly linking each finding back to the study's core questions (e.g., assessing H&E and AB/PAS as alternatives to Giemsa), and using more tempered language when discussing performance differences. In the discussion, this is where most of the overreach happen. The study do something useful, but it do not yet redefine practice. I would strongly encourage the authors to separate what the data directly show from what they hope might be true in future settings. There is a tendency to generalize beyond the cohort and beyond the technical scope, and pulling that back would actually make the paper more credible. A bit more self-critique, especially around generalizability and clinical translation, would be welcome. It almost get there, but not quite. Response As suggested, we have thoroughly revised the Discussion to address these points. We have focused on strengthening the self-critique regarding generalizability and clinical translation, ensuring that the interpretations are grounded explicitly in the data presented. Thank you. View more View less Competing Interests The authors declare no competing interests. reply Respond Report a concern Liu H. Peer Review Report For: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.5256/f1000research.187732.r435909) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-1148/v1#referee-response-435909 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gimiga N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 25 Nov 2025 | for Version 1 Nicoleta Gimiga , Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania 0 Views copyright © 2025 Gimiga N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript presents a large retrospective analysis comparing three widely used histological stains—H&E, Giemsa, and AB/PAS—for the detection of Helicobacter pylori in gastric biopsies. The topic is relevant, as histological diagnosis remains a cornerstone in H. pylori detection, and the study addresses a practical gap concerning diagnostic performance and cost-effectiveness. Strenghts: - Large sample size The inclusion of 816 biopsy cases is a major strength and provides statistical reliability and generalizability. Direct comparison of three staining methods -Few studies offer such a direct and comprehensive comparison. -Addresses a genuine gap in routine diagnostic pathology. Blind assessment- The use of three independent examiners helps minimize observer bias. LIMITATIONS: Retrospective design Use of Giemsa as the sole “gold standard” The manuscript is promising and clinically relevant , with strong methodological foundations and valuable practical implications. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Pediatrics, Pediatric gastroenterologyst I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Gimiga N. Peer Review Report For: Assessing the diagnostic accuracy of routine hematoxylin and eosin, Alcian Blue/Periodic Acid-Schiff, and Giemsa stains in the detection of Helicobacter pylori in gastric biopsies [version 1; peer review: 1 approved, 1 approved with reservations] . F1000Research 2025, 14 :1148 ( https://doi.org/10.5256/f1000research.187732.r429654) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-1148/v1#referee-response-429654 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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