Helicobacter pylori (H. pylori) is a highly prevalent infectious agent that affects ~40% of the global population (1). H. pylori was first discovered in 1983 by Robin Warren and Barry Marshall in gastric mucosal biopsy samples from patients with chronic active gastritis and peptic ulcer disease (2). Currently, this organism is recognized as the causative agent of gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric B-cell lymphoma (MALT lymphoma) (3).

Several in vivo and in vitro studies have demonstrated that H. pylori can invade epithelial cells and the lamina propria, triggering a more intense mucosal inflammatory response than bacterial attachment to epithelial cells alone. This process is important in the initiation and development of gastric inflammation (4-7). Furthermore, H. pylori that invades the gastric mucosa may translocate to gastric lymph nodes, leading to chronic immune system stimulation (8).

H. pylori in the lamina propria or subepithelial regions may be underdiagnosed without an appropriate immunohistochemical (IHC) study. Ito et al (8) found H. pylori in both the mucous and deeper layers of gastric tissue in patients with gastric cancer using the TMDU antibody. However, the study focused on cancerous conditions and did not provide further descriptions or categorizations of the distribution patterns of the bacteria.

Proton pump inhibitors (PPIs) are among the most frequently prescribed medications to alleviate dyspeptic pain and have demonstrated in vitro anti-H. pylori activity (9). These drugs can reduce the H. pylori load while inhibiting its urease activity (10,11) and may interfere with certain H. pylori detection tests, potentially leading to false-negative results (12).

Following PPI therapy, rod-shaped H. pylori can convert into its coccoid form (13,14). Research has indicated that coccoid H. pylori may contribute to bacterial transmission and the recurrence of infection, even following antimicrobial treatment (13-16). However, the exact mechanisms underlying the pathogenesis of coccoid H. pylori remain poorly understood.

The detection of coccoid H. pylori requires IHC staining because simple histochemical stains cannot reliably detect low numbers of coccoid or intracellular bacteria, nor can they differentiate coccoid H. pylori from artefacts or other bacteria. IHC stains offer high specificity, enabling accurate identification of H. pylori and exclusion of other similar-shaped organisms (17).

It was hypothesized that PPI treatment for acid suppression may modify the gastric mucosal microenvironment, influencing the distribution of H. pylori in both the surface and subepithelial compartments. Using IHC staining, it was aimed to identify distinct infection patterns and their potential associations with clinical outcomes.

The study protocol complied with the principles of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Navamindradhiraj University (approval no. 120/2566; Bangkok, Thailand). Gastric tissue biopsies from patients diagnosed with gastritis, who underwent endoscopic examination at Vajira Hospital (Faculty of Medicine, Navamindradhiraj University, Bangkok, Thailand) between October and December 2022, were reviewed retrospectively.

Inclusion criteria were as follows: i) Availability of at least four biopsies obtained from both the antrum and corpus as part of routine clinical practice; ii) diagnosis of gastritis confirmed by histopathological evaluation; and iii) sufficient formalin-fixed paraffin-embedded (FFPE) tissue for both hematoxylin and eosin (H&E) and IHC analysis. Additional exclusion criteria included: vii) inadequate biopsy samples, and viii) poor tissue preservation precluding histological interpretation.

As part of standard clinical practice, at least four biopsies were collected from both the gastric antrum and corpus. Patients were not specifically recruited for the present study, and no additional tissue samples were collected. FFPE blocks were processed for histopathological and IHC analysis. Clinical data were also extracted and recorded between April and May 2024. All identifying information was removed.

The patients were divided into two groups according to PPI therapy: Those receiving PPIs for at least a 14-day period prior to the procedure (PPI-treated group) and those not receiving PPIs or receiving them for a shorter period (PPI-untreated group).

In the present study, the prevalence of H. pylori infection, as determined by IHC in 255 patients, was 32.5%, which is lower than the 40-45% reported in previous studies (1,20,21). This discrepancy may be attributed to differences in study periods, with more recent research indicating a decline in prevalence compared with earlier studies (1,20,21). This reduction in infection rates could be due to demographic variations, improved personal hygiene, improved living conditions, increased public awareness, and active H. pylori eradication efforts (1,20,21).

The ability to detect H. pylori and its specific localization may vary depending on the antibody used for IHC staining. Certain antibodies typically detect H. pylori only in superficial areas and not beneath the epithelium. Ito et al (8) examined the presence and invasive behavior of H. pylori via various methods, including IHC, PCR, bacterial culture and immunoelectron microscopy. They found H. pylori in both the mucous layer and the lamina propria in gastric cancer patients using the TMDU-mAb. In the present study, the diagnostic accuracy of BioGenex was comparable with that of TMDU, with BioGenex showing greater sensitivity in detecting H. pylori in deeper tissue, where TMDU was detected in only 68.9% of cases.

H. pylori was initially considered a non-invasive pathogen that resides solely in the gastric lumen and affects only gastric epithelial cells. However, most previous studies focused primarily on the surface epithelium. Subsequent in vivo and in vitro studies have shown that H. pylori is invasive and can also be found in the lamina propria (4-8). Expanding the focus to include the subepithelial location may provide a more accurate representation of H. pylori prevalence.

The presence of H. pylori in the subepithelial region correlated more strongly with the severity of chronic inflammation than surface bacteria in active gastritis, which aligns with the findings of Ito et al (8). This suggests a location-dependent immunological response, potentially involving macrophage interaction and contributing to the induction and maintenance of chronic inflammation in H. pylori-infected gastric mucosa. While surface epithelial H. pylori typically induces IL-12 production and Th1 cell accumulation (22), subepithelial bacteria, interacting with lamina propria macrophages and T cells, may elicit a distinct chronic inflammation. The present study observed a significantly higher prevalence of isolated subepithelial H. pylori (27.7%) compared with Ito et al's (2%). Notably, Ito et al (8) reported 46 PCR-positive H. pylori cases in the stomach; however, 4 of these cases exhibited negative IHC results for both surface and subepithelial bacteria, with only 1 showing isolated subepithelial positivity. The differences in findings may be explained by several factors. Firstly, Ito et al (8) employed more thorough tissue sampling, which may have led to an increased detection of surface bacteria.

In contrast to surface-attached bacilli, subepithelial H. pylori presented as dot-like signals, which may represent bacterial casts or coccoid forms. Additionally, the enhanced sensitivity of BioGenex used in the present study may explain the discrepancies. BioGenex utilizes the ULC3R clone to identify specific epitopes of H. pylori that are resistant to intracellular digestion and capable of detecting antigenic forms of H. pylori resembling the bacillary type, even in coccoid forms. BioGenex detected all TMDU-positive cases in the subepithelium, indicating greater sensitivity. Furthermore, differing positivity cutoffs (1 in the present study vs. ≥10 signals in Ito et al's) could contribute to the observed variation. Consequently, the precise prevalence of isolated subepithelial H. pylori remains inconclusive, necessitating further validation.

A significantly lower detection rate of H. pylori in the subepithelial compartment was observed in patients treated with PPIs than in those not treated with PPIs. These findings suggest that PPI use may inhibit H. pylori infiltration into the lamina propria.

The underlying mechanism may involve inhibiting MMP-2/TIMP-3 interactions. These effects could protect against gastric mucosal injury, thereby reducing the degree of epithelial damage that facilitates bacterial translocation (23).

In addition to the diagnostic challenges of detecting subepithelial bacteria, their presence may have implications for clinical outcomes. First, subepithelial particles could signify residual bacteria from the surface that remain viable in unbiopsied areas of the stomach, potentially causing persistent symptoms. Second, immunoreactive signals in subepithelial regions may represent debris from dead bacteria, which can continue to trigger inflammation and contribute to ongoing inflammatory responses.

Third, subepithelial immunoreactive particles may reflect a transformation from the usual bacillary form of H. pylori to an atypical coccoid form under stressful conditions. These coccoid forms could revive and revert to culturable bacilli. This transformation may contribute to treatment failure and relapse, as coccoid forms may evade immune detection while remaining susceptible to antibiotics (13-16). These findings may explain our results, which revealed that the eradication rate of H. pylori treatment was greater in patients with isolated surface epithelial or mixed infection patterns than in those with an isolated subepithelial pattern. The presence of the latter pattern may provide indirect evidence supporting the consideration of a step-up eradication therapy regimen. However, establishing a definitive link between the isolated subepithelial pattern and treatment success or failure remains challenging due to the small sample size.

Unlike treated patients, most untreated H. pylori-positive patients exhibited an isolated subepithelial pattern, likely due to underdiagnosis. Most treated patients are diagnosed using the rapid urease test (RUT), H&E staining, Giemsa, or polyclonal DAKO IHC, which are routinely employed in Vajira Hospital. By contrast, untreated patients predominantly presented with an isolated subepithelial pattern, which typically yields negative results for H&E, Giemsa, or polyclonal DAKO IHC. A total of 5 patients were treated on the basis of positive RUT results. However, RUT is rarely performed at our institution; treatment decisions are typically based on histopathology (H&E), special stains, or IHC results. This underscores the potential for significant underdiagnosis in real-world clinical practice.

Whether this staining represents live subepithelial bacteria or merely debris from dead bacteria remains unclear. Nevertheless, our results suggest that this immunoreactive signal should not be overlooked and warrants further investigation.

The current reporting system for H. pylori may benefit from the incorporation of immuno-stained subepithelial bacteria as a prognostic factor. The authors propose reporting H. pylori-IHC as an ancillary study alongside the Updated Sydney System to provide a clearer summary of H. pylori density and improve clinical communication. Specifically, infection patterns should be reported under the heading ‘H. pylori-positive cases’, including specific location details. This would enable clinicians to accurately identify infection types and guide treatment decisions. The isolated subepithelial pattern may suggest the necessity for step-up therapy, closer monitoring, or additional testing, such as urea breath tests or stool H. pylori antigen tests, in accordance with current guidelines (24). Using IHC, H. pylori density grading can be easily summarized on the basis of the groups described in the methodology for clinical implications.

The overall pattern should be reported under the heading ‘H. pylori-positive cases’ followed by a description of the specific location. This approach would improve communication with clinicians, for example, ‘isolated surface epithelial pattern (surface bacteria 3, negative subepithelial signal)’ or ‘non-specific pattern (surface bacteria 3, subepithelial signal 2)’. This pattern group system aims to improve description of H. pylori behavior and could guide further management, particularly for the isolated subepithelial pattern group. In such cases, patients should undergo a urea breath test or a stool H. pylori antigen test, according to guidelines. The presence of this pattern suggests that step-up therapy and close follow-up or further investigation should be considered. By contrast, the surface-attached groups had a greater likelihood of successful treatment than did the isolated subepithelial pattern group.

Although the present study provides valuable insights into H. pylori, it has several limitations. The primary limitations are its retrospective design and the small number of patients with complete clinical follow-up data, which may affect the generalizability of the findings. Additionally, since only omeprazole is included in the national reimbursement program, other PPIs could have varying effects on the outcomes. Further prospective studies with larger cohorts and broader medication coverage are needed to validate and expand upon these findings.

In conclusion, H. pylori, particularly in subepithelial locations, can be underdiagnosed even with IHC tests. The detection rate is influenced by the sensitivity of the primary antibody used and the location of the bacteria. The effect of PPIs might prevent surface bacterial translocation. This feature should be included in pathological assessments and reports as an ancillary study alongside the updated Sydney System. Subepithelial H. pylori infection could serve as an independent prognostic factor for clinical outcomes and could guide further patient management.