H. pylori infection is associated with several gastrointestinal complications, including mild chronic gastritis, gastric carcinoma, and gastric lymphoma. The accurate diagnosis of H. pylori infection is important to devise an effective treatment for these gastric complications. Among the several diagnostic methods developed for detecting H. pylori, direct detection of H. pylori in the gastric mucosa is the most accurate method.
This study aimed to evaluate the efficacy of IHC staining to directly detect H. pylori in the gastric biopsies with minimal and/or atypical infection obtained from 50 Saudi patients with chronic gastritis. The diagnostic accuracies of IHC staining were compared with those of routine H&E staining, modified Giemsa staining, and qRT-PCR, which was considered as the diagnostic gold standard in this study.
Previous studies have reported that qRT-PCR analysis is one of the most sensitive diagnostic tools for the detection of H. pylori [13–18]. The high specificity of qRT-PCR analysis is due to the use of specific primers and internal controls, which minimise false-positive results. Additionally, the ability of qRT-PCR to detect low counts of H. pylori minimises false-negative results. In this study, H. pylori detection was performed using primer-probe-based qRT-PCR targeting the RNA polymerase beta-subunit (rpoB) gene of H. pylori along with suitable internal controls that ensured high specificity and sensitivity.
The qRT-PCR analysis of 50 biopsy specimens revealed an H. pylori infection incidence rate of 64%, which was higher than that in other studies, which reported H. pylori infection prevalence of 48.7% and 57% [19, 20]. Previous studies in Saudi Arabia have reported that the H. pylori infection prevalence rates ranged from 28–70% with an average of 50% . The variation in H. pylori prevalence rates among different studies can be attributed to differences in patient characteristics, administration of antibiotic therapy, sampling conditions, and qRT-PCR modality.
The false-positive and false-negative rates for routine H&E staining were 16 and 26%, respectively. The high false-positive rates associated with H&E staining can be attributed to the inability of this method to differentiate H. pylori from the surrounding gastric mucosa. Additionally, the inability of H&E staining to differentiate H. pylori from gastric secretions or eosinophilic debris may contribute to high false-positive rates. Of the 50 cases, modified Giemsa staining and H&E staining identified 21 (42%) and 27 (54%) H. pylori-positive cases, respectively. The specificity of modified Giemsa staining (61.11%) for H. pylori detection was higher than that of H&E staining (55.56%); this concurred with the results of Laine et al . In addition to uniform staining, modified Giemsa staining enables the identification of distinctive morphologies of H. pylori. However, the sensitivity of modified Giemsa staining for the detection of H. pylori was lower than that of H&E staining. The degree of infection is reported to affect the sensitivity of modified Giemsa staining . The low sensitivity of modified Giemsa staining can be attributed to the inability of this method to distinguish H. pylori adhering to the glandular epithelium, especially in cases with mild degree of colonisation. Both H. pylori and glandular epithelium appear blue upon Giemsa staining. H. pylori transforms its morphology from spiral form to atypical forms under harsh conditions, such as antibiotic treatment. These atypical forms of bacteria cannot be identified through modified Giemsa staining, which may contribute to low sensitivity of modified Giemsa staining.
The sensitivity (87.5%) and specificity (94.44%) of IHC staining were higher than those of H&E and modified Giemsa staining, and these values concurred with the results of previous studies [10, 23, 24]. In contrast to H&E staining, the histopathological changes cannot be evaluated through IHC staining. However, IHC staining is considered the most accurate direct histopathological staining method for the detection of H. pylori. The high sensitivity and specificity of IHC staining are due to the use of specific antibodies, ability to detect atypical bacterial forms, such as coccoid forms, and low false-positive rates. Moreover, IHC is recommended when other routine methods fail to detect H. pylori in cases of chronic gastritis caused due to minimal infection or atypical distribution of bacteria in affected tissue . The limitation of IHC staining for routine diagnostic application is high cost. Hence, IHC staining is recommended for cases with minimal H. pylori infection .
In this study, H&E staining revealed that 12 (24%), 24 (48%), and 14 (28%) patients exhibited mild, moderate, and severe chronic gastritis, respectively. Of the 27 H. pylori-positive cases identified through H&E staining, 35%, 60%, and 5% cases exhibited mild, moderate, and severe infections, respectively. These results concurred with those of Siddiqui et al  who reported 45 (24.4%), 137 (74%), and 3 (1.6%) cases of mild, moderate, and severe chronic gastritis, respectively, among 185 chronic gastric cases, with H. pylori-positive rates of 17.5%, 80.7%, and 1.8%, respectively. In this study, H. pylori was majorly associated with moderate inflammation. The section must be carefully examined for the presence of H. pylori in cases of moderate inflammation associated with active chronic gastritis. Previous studies have reported the correlation between chronic active gastritis and presence of H. pylori infection [28–30].
In this study, gastric ulcer was detected in 30% of cases. H. pylori is reported to be the major risk factor for the development of gastric ulcers as more than two-thirds of patients with gastric ulcer test positive for H. pylori infection . Additionally, H. pylori infection is an important risk factor for the development of gastric atrophy and IM [32–34]. This is consistent with the results of this study in which all four detected IM cases were H. pylori-positive. Previous studies have reported that the presence of lymphoid follicles in the gastric mucosa is a common response to H. pylori infection and that the lymphoid follicles are detected in the gastric mucosa in 27–100% of cases [29, 35]. In this study, 22 (44%) out of the 50 studied cases were associated with lymphoid follicles with or without germinal centres and all 22 cases were H. pylori-positive. The presence of lymphoid follicles in the gastric mucosa appears to be a strong predictor of H. pylori infection. These results were consistent with those of Afzal et al.  who examined 500 cases of chronic gastritis and reported that 95.8% of cases with follicular gastritis tested positive for H. pylori. Moreover, another study examining the histopathology of gastric biopsies reported that the presence of lymphoid follicles indicates high probability of H. pylori infection . Under physiological conditions, the histological structure of stomach does not contain lymphoid follicles . Hence, the presence of H. pylori-associated lymphoid tissue is considered as an important pathological feature in the stomach. The development of mucosa-associated lymphoid tissue (MALT) precedes the development of primary MALT lymphoma.