**2. Materials and methods**

#### **2.1. Patients**

This was a prospective, single-centre study (in Maria Sklodowska Curie Children's Emergency Hospital Bucharest, Romania) that evaluated consecutive children referred by their physicians for an upper endoscopy because of dyspepsia. They were all screened for *H. pylori* and presented a positive stool antigen test.

Excluding criteria were the use of proton pump inhibitors or H2-receptor antagonists and antibiotics as well as non-steroidal anti-inflammatory drugs or steroidal treatment 2 weeks before the beginning of the study, previous intestinal surgery (except for polypectomy and appendectomy), concomitant severe disease (heart, lungs, kidney and endocrine diseases) and smoking or alcohol consumption among adolescents.

The study was approved by the ethics committee.

## **2.2. Endoscopy**

the stomach and duodenum and inducing a systemic humoral immune response [1]. *H. pylori* survival in the acidic gastric environment is mediated by mechanisms such as activity of the urease enzyme, which catalyses and hydrolyses urea to form carbon dioxide and ammonia, producing a neutral environment that is essential for its survival. The primary routes of transmission are considered to be faecal-oral and oral-oral, but some indirect evidences report that the infection can also be acquired by drinking water and by other environmental sources [2, 3]. *H. pylori* represents a key factor in the aetiology of various gastrointestinal diseases, ranging from asymptomatic chronic active gastritis to peptic ulceration, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Other diseases caused by the pathogen are iron deficiency anaemia, chronic idiopathic thrombocytopenic purpura and growth retardation. *H. pylori* was classified as a class I human carcinogen by the World

Numerous diagnostic tests are available for detecting *H. pylori* infection: invasive techniques, which means endoscopy with biopsies for a rapid urease test (RUT), histology and culture and noninvasive techniques, such as serology, 13C-Urea breath test (13C-UBT) and stool antigen test. There is no single method to detect *H. pylori* infection reliably and accurately. The choice of the diagnostic method depends on patients' age and complaints, technical difficulty

Two tests are recommended to define *H. pylori* status in children: bacterial culture of gastric biopsy and histology [4]. Bacterial culture of gastric biopsy has 100% specificity, but its sensitivity is low. *H. pylori* can be cultured from gastric biopsies, although this method often presents some difficulties. *H. pylori* soon loses viability when exposed to the environment, and biopsies should be cultured quickly. If it is not possible, a transport media may be used. Histology provides an excellent diagnostic accuracy, allowing for the detection of the bacteria as well as for the grading of gastritis. The sensitivity and specificity of histology for the diagnosis depend

The aims of our study were to assess the histological findings and to compare them with the results of bacterial cultures, obtained through gastric biopsy, in children with *H. pylori* gastritis. We also wanted to find out the possible factors that may influence bacterial culture outcomes.

This was a prospective, single-centre study (in Maria Sklodowska Curie Children's Emergency Hospital Bucharest, Romania) that evaluated consecutive children referred by their physicians for an upper endoscopy because of dyspepsia. They were all screened for *H. pylori* and

Excluding criteria were the use of proton pump inhibitors or H2-receptor antagonists and antibiotics as well as non-steroidal anti-inflammatory drugs or steroidal treatment 2 weeks

on clinical settings, density of colonisation and experience of the histopathologist [5].

Health Organization in 1994.

80 Histology

**2. Materials and methods**

presented a positive stool antigen test.

**2.1. Patients**

level, costs and extensive accessibility in hospitals.

All patients underwent endoscopy with biopsy specimens for histology (one for the antrum, one for the corpus). One sample from the antrum was used for rapid urease test. Two additional biopsies were taken from the antrum for bacterial culture. The samples were placed into separate vials, previously identified, containing the appropriate medium for each test. The first sample was used for bacterial culture.

This procedure was performed in patients with a minimum of 10 hours of fasting, under general anaesthesia or conscious sedation. Vital signs were continuously monitored for the entire procedure.

Written informed consent was obtained from the parent or guardian of each child included in the study.

#### **2.3. Bacterial culture**

The biopsy specimens collected for bacterial culture were transported in commercial selective transport *H. pylori* medium, Portagerm pylori (BioMérieux SA, Marcy l'Etoile, France), and were inoculated after a few hours onto selective medium pylori agar (BioMérieux Italia). The plates were incubated under microaerobic condition at 37° for 72 hours. Once incubated, the colonies resembling *H. pylori* were identified by Gram stain and by oxidase, catalase and urease tests. Suspensions from the primary plates were prepared in sterile solution to perform an E-test on pylori agar. An agar plate was streaked in three directions with a swab dipped into each bacterial suspension to produce a lawn of growth; an E-test strip (E-test; AB Biodisk, Solna, Sweden) was placed each onto separate plates, which was immediately incubated in a microaerobic atmosphere at 37° for 72 hours. Isolated strains were tested for amoxicillin, clarithromycin, metronidazole and levofloxacin resistance following the recommendations of the European Committee on Antimicrobial Susceptibility Testing.

#### **2.4. Histology**

A biopsy of the gastric body and antrum was fixed in a solution of formaldehyde 10%. Subsequently, the gastric mucosa samples were processed, following the usual steps of dehydration and paraffin embedding.

Two stains were used for histological study: haematoxylin-eosin and Giemsa. Haematoxylin-eosin stain was used to evaluate inflammatory cells and *H. pylori* (**Figure 1**). Giemsa stain was needed when haematoxylin-eosin stain failed to identify the bacterium (**Figure 2**). Giemsa stain is the preferred stain for detecting *H. pylori* because of its technical simplicity, high sensitivity and low cost.

deviation (SD). Differences and relationships between variables were analysed using Fisher's exact for low expected frequencies. A p < 0.05 was considered statistically significant for all

A Study of the Correlation between Bacterial Culture and Histological Examination in Children…

http://dx.doi.org/10.5772/intechopen.80257

83

We calculated the sensitivity and specificity for *H. pylori* culture and histology. Sensitivity and specificity may be defined, respectively, as the probability of having a positive test in a person with the disease (sensitivity) and the probability of having a negative test in a person without

In the study, the culture findings and histological examination findings were accepted as "gold standard". The detection of *H. pylori* in at least one of the two tests was accepted as *H. pylori* positivity. Negative results in both culture and histology were accepted as *H. pylori*

Of the 38 patients who underwent upper endoscopy with biopsies by protocol (**Figure 3**), nine

Twenty-nine cases (76.31%) were included in the final analysis, nineteen females (65.51%) and ten males (34.49%). The ages were between 3 years and 7 months and 17 years and 8 months

The results for the diagnosis of *H. pylori* infection for each of the tests revealed that the haematoxylin-eosin and Giemsa stains of the antrum and body were the test that identified a higher

Indeed, the histological examination of samples was able to identify the presence of *H. pylori* in 28 patients (96.55%), while the culture resulted to be positive in only six cases (21.42%).

We did not analyse separately the presence of the *H. pylori* in the antral mucosa compared with the gastric body mucosa; the result was recorded positive, if the bacterium was isolated

The histology also showed that 14/28 (50%) patients had mild *H. pylori* density, 11 (38.29%)

In one case, the culture was positive, but the bacterium was not identified through the histological exam. Among the other five cases with positive culture, two were associated with a mild score of *H. pylori* density, the other two with a moderate score and one with a marked

We analysed the correlation between densities of *H. pylori* in histological exam and positive *H. pylori* culture: 14.28% (2/14) of patients with mild *H. pylori* density, 18.18% (2/11) of those with moderate density and 33.33% (1/3) of those with marked density had positive results in bacterial culture. There was not a statistically significant correlation between the degree of *H. pylori* density observed at histology and the positive result of bacterial culture (p *=* 0.7). The limited number of patients with positive bacterial culture may have influenced these

were excluded because of negative results in both culture and histology.

number of *H. pylori* infection than the *H. pylori* culture.

had moderate density and 3 (10.71%) had marked density.

the analysed parameters.

the disease (specificity).

(mean age 13, 5 ± 4.53 years).

in any of the histological examinations.

**3. Results**

negativity.

score (**Table 1**).

results.

**Figure 1.** *Helicobacter pylori* in histological section of the gastric mucosa stained with haematoxylin-eosin. This figure represents a 200× histological section of haematoxylin-eosin-stained gastric mucosa. It is seen with diffuse inflammatory lymphoplasmacytic infiltrate (black arrows) and vascular congestion (red arrows). *Helicobacter pylori* is found at the surface of the gastric mucosa within the layer of mucus (yellow arrows).

**Figure 2.** *Helicobacter pylori* in histological section of the gastric mucosa stained with Giemsa. This is a 400× histological section of Giemsa-stained gastric mucosa biopsy. There can be seen colonisation with *Helicobacter pylori* (white arrows) of the gastric glands, represented by small curved structures.

Gastritis was graded according to the Sydney system [6] that assesses the severity of inflammation, the level of activity (the degree of polymorph neutrophil inflammation) and the presence of atrophy and of intestinal metaplasia on a scale from 0 to 3.

In accordance with the Sydney system, the density of *H. pylori* infection was also graded semiquantitatively on a scale from 0 to 3 (mild, moderate and marked).

*H. pylori* was recognised in the histological section appearing as a short-curved or spiral bacillus resting on the epithelial surface or in the mucus layer.

#### **2.5. Statistical analysis**

The data was collected and analysed with Microsoft Excel 2013 and PSPP version 1.0.1. Continuous variables with a normal distribution were expressed as a mean with standard deviation (SD). Differences and relationships between variables were analysed using Fisher's exact for low expected frequencies. A p < 0.05 was considered statistically significant for all the analysed parameters.

We calculated the sensitivity and specificity for *H. pylori* culture and histology. Sensitivity and specificity may be defined, respectively, as the probability of having a positive test in a person with the disease (sensitivity) and the probability of having a negative test in a person without the disease (specificity).
