Comparison of special stains (Giemsa stain and Modified Toluidine Blue stain) with immunohistochemistry as gold standard for the detection of H. pylori in gastric biopsies

Helicobacter pylori (H. pylori) are spiral shaped, curved micro-aerophilic gram-negative bacilli that are most frequently found in routine gastric biopsies of patients with chronic gastritis. Chronic gastritis affects more than 50% of the world’s population [1]. Despite medical advancements, H. pylori infection is still widely prevalent, especially in Asia [2], and is quite rampant in developing countries including Pakistan.

According to a regional study by Rasheed et al., in which 93 adult patients with upper gastrointestinal symptoms underwent endoscopic biopsy, H. pylori infection was confirmed by routine histopathology, rapid urease test, and culture. The frequency of infection calculated in males and females was 57.7% and 72.7%, respectively [3].

The role of H. pylori in the etiology of chronic gastritis, peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma (MALT) is well known [4]. Recent literature reveals an association of this bacterium with many extragastric diseases including iron deficiency anemia, idiopathic thrombocytopenic purpura, rosacea, chronic prurigo, pre-eclampsia, chronic cholecystitis, diabetes mellitus, and Grave’s disease, among others [5].

H. pylori is also associated with many malignancies, namely, laryngeal squamous cell carcinoma [6], diffuse large B cell lymphoma [7], and colorectal cancer [8]. To establish the primary diagnosis and posttreatment confirmation of H. pylori eradication, several invasive and noninvasive diagnostic tests are used. Invasive tests include culture, endoscopy, histological and immunohistochemical staining of biopsied tissue, rapid urease test, and PCR [9], whereas noninvasive tests include serological testing by ELISA, urea breath test, and fecal antigen test [10]. Among these, histological examination of tissues obtained by endoscopic biopsy is one of the most useful diagnostic tools for H. pylori detection [11].

Several histochemical stains including H&E, Giemsa, Warthin–Starry, Gimenez, Genta, and Toluidine blue as well as immunohistochemistry (IHC) using an H. pylori antibody have been applied to detect H. pylori in gastric biopsies. Many pathologists affirm that H&E-stained sections are sufficient for the diagnosis of H. pylori [12]. However, H&E staining presents little contrast between tissue and bacteria, which increases the difficulty of the assessment by the pathologist and increases the time per case for evaluation of gastric biopsy specimens (Fig. 1). Additional stains are required in cases of atrophic mucosa, mild inflammation, and post eradication therapy [13]. Among the ancillary stains, Giemsa stain is the most sensitive, easy to perform, and reproducible and is popular worldwide for the diagnosis of H. pylori-induced gastritis [14]. Recent literature reveals that Modified Toluidine Blue stain (MTB) is superior to H&E and is advantageous for use in developing countries in terms of cost, availability, and gastric cancer screening [15].

IHC, which is more specific and is associated with less interobserver variation, has an advantage in cases of low-grade gastritis and infection by coccoid bacterial forms. IHC has been approved as a reliable method by multiple authors and is routinely applied internationally as the method of choice for the diagnosis of H. pylori infection [16], [17], [18]. However, in developing countries, it is not financially feasible to use IHC for all routine gastric biopsies, and the immunohistochemistry technique is not widely available except in large laboratory setups.

The probability of detecting H. pylori in normal gastric mucosa is extremely low. To optimize the detection of this bacterium, the updated Sydney System protocol of obtaining 5 endoscopic biopsy punches (two each from the antrum and corpus and one from the incisura angularis) is universally accepted. Ancillary histochemical stains enhance detection in cases of lymphocytic gastritis, granulomatous gastritis, eosinophilic gastritis, chronic active gastritis (if H&E fails to demonstrate H. pylori), and chronic inactive gastritis (in the presence of gastroduodenal ulcer, MALT lymphoma/adenocarcinoma, and duodenal lymphocytosis as well as after H. pylori treatment and in high-risk demographic/low socioeconomic areas) [19].

In our clinical practice, only H&E staining was applied for the analysis of gastric biopsies. This study was novel in our region, and because of the high prevalence of H. pylori in Pakistan and its grave outcomes, the need to provide healthcare institutions with diagnostically accurate and cost-effective stains to identify this bacterium is urgent. The first aim was to determine the rational use of the appropriate histochemical stains in our histopathology practice and to suggest an easily accessible stain with diagnostic capability that is close to IHC and that can be routinely applied in low-resource settings without a lag of diagnostic yield. For this purpose, H&E, Giemsa, and MTB stains were compared with each other and verified against IHC. The second objective was to evaluate whether MTB can be more reliable than Giemsa for H. pylori diagnosis.

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