**2. Material and methods**

#### **2.1. Patients**

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

Demographic characteristics and family history of each patient were collected through a questionnaire, which was completed by parents or by patients depending on the age of the child. Demographic data included patients' age, gender, and residency (urban or country area). Information on patient's history of *H. pylori* infection as well as on previous therapies was obtained. History of siblings or parents infection was also assessed. Patients were asked about the time of onset and duration of gastrointestinal symptoms, use of proton pump inhibitors, H2 receptors antagonists, non steroidal anti-inflammatory drugs or steroidal drugs. Smoking status and alcohol consumption was determined as well.

Excluding criteria were: use of proton pump inhibitors or H<sup>2</sup> receptors antagonists and antibiotics as well as non steroidal anti inflammatory drugs or steroidal therapy 2 weeks before the beginning of the study, history of intestinal surgery (except for polypectomy and appendectomy), concomitant severe disease (heart, lungs, kidneys and endocrine diseases), and smoking and alcohol consumption.

The study was approved by Ethics Committee.

#### **2.2. Endoscopy**

The public health importance of *H. pylori* discovery, in 1982, and its role in stomach disease was recognized in 2005 with the attribution of the Nobel Prize in Medicine to Barry Marshall and Robin Warren. *H. pylori* was classified as a class I human carcinogen by World Health

Numerous diagnostic tests are available for detecting *H. pylori* infection: invasive techniques, which means endoscopy with biopsies for a rapid urease test (RUT), histology, culture and non-invasive techniques, such as serology, 13C-Urea breath test (13C-UBT), and the 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

The same diagnostic methods used for adults can be used for children. However, *H. pylori* infection has certain particularities in children which have implications for diagnostic testing. *H. pylori* infection may slowly establish itself, so it is possible, in rare instances, to find the bacteria without traces of inflammation. At endoscopy, antral nodularity is common [2]. 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 depends on clinical settings, density of colonization and on pathologist's experience [3].

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

Demographic characteristics and family history of each patient were collected through a questionnaire, which was completed by parents or by patients depending on the age of the child. Demographic data included patients' age, gender, and residency (urban or country area). Information on patient's history of *H. pylori* infection as well as on previous therapies was obtained. History of siblings or parents infection was also assessed. Patients were asked about the time of onset and duration of gastrointestinal symptoms, use of proton pump inhibitors,

receptors antagonists, non steroidal anti-inflammatory drugs or steroidal drugs. Smoking

biotics as well as non steroidal anti inflammatory drugs or steroidal therapy 2 weeks before the beginning of the study, history of intestinal surgery (except for polypectomy and appendectomy), concomitant severe disease (heart, lungs, kidneys and endocrine diseases), and

receptors antagonists and anti-

Organization in 1994.

**2. Material and methods**

a positive stool antigen test.

smoking and alcohol consumption.

The study was approved by Ethics Committee.

**2.1. Patients**

H2

level, costs and extensive accessibility in hospitals.

36 Helicobacter Pylori - New Approaches of an Old Human Microorganism

status and alcohol consumption was determined as well.

Excluding criteria were: use of proton pump inhibitors or H<sup>2</sup>

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 in separates vials, previously identified, containing the appropriate medium for each test.

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

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

#### **2.3. Histology**

A biopsy of gastric body and antrum were 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: hematoxylin eosin and Giemsa. Hematoxylin eosin stain was used to evaluate inflammatory cells and *H. pylori*. Giemsa stain was needed when hematoxylin eosin stain failed to identify the bacterium. The Giemsa stain is the preferred stain for detecting *H. pylori* because of its technical simplicity, high sensitivity and low cost.

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 semi quantitatively classified on a scale from 0 to 3 (mild, moderate, and marked).

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

#### **2.4. 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 h. 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 Bio disk, Solna, Sweden) was placed each onto separate plate, which was immediately incubated in a microaerobic atmosphere at 37°C for 72 h. Isolated strains were tested for amoxicillin, clarithromycin, metronidazole, and levofloxacin resistance following the recommendations of the European Committee on Antimicrobial Susceptibility Testing.

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

The data was collected and analyzed with Microsoft Excel 2013 and PSPP version 1.0.1. Continuous variables with a normal distribution were expressed as a mean with standard derivation (SD) and continuous variables with a non-normal distribution as median with interquartile range (IQR). Differences between groups were analyzed using Student t-test and Mann-Whitney U test for continuous variables, and Fisher's exact test for categorical variables. A p value <0.05 was considered statistically significant for all the analyzed parameters.

#### **3. Results**

Of the 38 patients who underwent upper endoscopy with biopsies by protocol (**Figure 1**), nine were excluded because of negative results in both culture and histology.

In the study, the culture and histology 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* negativity.

Twenty-nine cases (76.31%) were included in the final analyses, 19 females (65.51%) and the 10 males (34.49%). The ages were between 3 years and 7 months and 17 years and 8 months (mean age 13.5 ± 4.53 years).

Four patients had a family history of peptic ulcer disease. In 15 children the duration of symptoms was more than 6 months and 12 patients were previously treated for *H. pylori* (**Table 1**).

The mean duration of the period between the onset of symptoms and the effective diagnosis in patients with a family history of upper gastrointestinal diseases was 3.75 ± 3.69 and 8.66 ± 5.42 months in those with negative family history (p = 0.17). A family history of gastric or duodenal ulcer did not significantly alter the length of time between the onset of symptoms and the diagnosis according to our statistical results, which, however, may have been influenced by the restricted number of patients in our study population.

Twelve patients had previous therapies. The median age of patients who were previously treated was 14.5 ± 3.74 and 13 ± 4.71 years old of those without any anterior therapy (p = 0.2).

The most common finding identified at endoscopy was macroscopic nodular antral gastritis, which was present in 22 patients (75.86%) (**Figure 2**). Among these, 10 had additional associated macroscopic lesions: 8 presented with nodular gastritis of gastric body, 1 with bulbitis, and one with esophagitis. Endoscopy showed antral hyperemia in 4 cases and a normal mucosal aspect in other 3 cases (**Table 2**).

We tried to find out if there was a significant difference in the severity of endoscopic findings between patients who received previous therapy and those who did not. Among the 12 previously treated patients, 7 (58.33%) presented with macroscopic nodular antral gastritis,

Endoscopical Aspects of *Helicobacter pylori* Gastritis in Children

http://dx.doi.org/10.5772/intechopen.81437

Mean age ± SD, years 13.5 ± 4.47 Male/female 10/29 Familial history for *H. pylori* infection 4/29 Peptic ulcer/non ulcer dyspepsia 1/28 Previous therapy 12/29

**Table 1.** Clinical and demographical characteristics of the patients.

**Figure 1.** Flow chart of the study.

**Figure 1.** Flow chart of the study.

in a microaerobic atmosphere at 37°C for 72 h. Isolated strains were tested for amoxicillin, clarithromycin, metronidazole, and levofloxacin resistance following the recommendations

The data was collected and analyzed with Microsoft Excel 2013 and PSPP version 1.0.1. Continuous variables with a normal distribution were expressed as a mean with standard derivation (SD) and continuous variables with a non-normal distribution as median with interquartile range (IQR). Differences between groups were analyzed using Student t-test and Mann-Whitney U test for continuous variables, and Fisher's exact test for categorical variables. A p value <0.05 was considered statistically significant for all the analyzed

Of the 38 patients who underwent upper endoscopy with biopsies by protocol (**Figure 1**), nine

In the study, the culture and histology 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* negativity.

Twenty-nine cases (76.31%) were included in the final analyses, 19 females (65.51%) and the 10 males (34.49%). The ages were between 3 years and 7 months and 17 years and 8 months

Four patients had a family history of peptic ulcer disease. In 15 children the duration of symptoms was more than 6 months and 12 patients were previously treated for *H. pylori* (**Table 1**). The mean duration of the period between the onset of symptoms and the effective diagnosis in patients with a family history of upper gastrointestinal diseases was 3.75 ± 3.69 and 8.66 ± 5.42 months in those with negative family history (p = 0.17). A family history of gastric or duodenal ulcer did not significantly alter the length of time between the onset of symptoms and the diagnosis according to our statistical results, which, however, may have been influ-

Twelve patients had previous therapies. The median age of patients who were previously treated was 14.5 ± 3.74 and 13 ± 4.71 years old of those without any anterior therapy (p = 0.2). The most common finding identified at endoscopy was macroscopic nodular antral gastritis, which was present in 22 patients (75.86%) (**Figure 2**). Among these, 10 had additional associated macroscopic lesions: 8 presented with nodular gastritis of gastric body, 1 with bulbitis, and one with esophagitis. Endoscopy showed antral hyperemia in 4 cases and a normal

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

enced by the restricted number of patients in our study population.

of the European Committee on Antimicrobial Susceptibility Testing.

38 Helicobacter Pylori - New Approaches of an Old Human Microorganism

**2.5. Statistical analysis**

parameters.

**3. Results**

(mean age 13.5 ± 4.53 years).

mucosal aspect in other 3 cases (**Table 2**).


**Table 1.** Clinical and demographical characteristics of the patients.

We tried to find out if there was a significant difference in the severity of endoscopic findings between patients who received previous therapy and those who did not. Among the 12 previously treated patients, 7 (58.33%) presented with macroscopic nodular antral gastritis,

**Figure 2.** Endoscopical aspect of *Helicobacter pylori* gastritis in children (macroscopic nodular antral gastritis).

**4. Discussion**

nodularity

therapies.

Antral hyperemia without macroscopic

Total 17 (100)

ulcers [6].

metastases or sites of MALT lymphoma resolve [4].

**Endoscopic features** *H. pylori* **infection without** 

Macroscopic nodular antral gastritis 15 (88.24) 7 (58.33%)

Esophagitis (with) 1 2 (16.67%)

Normal 0 12 (100%)

Nodular antral gastritis (only) 8 4 Nodular gastritis of corpus (with) 6 2 Erosive bulbitis (with) 0 1

There is a clear association between *H. pylori* and gastritis, gastric ulcer, and duodenal ulcers. Studies have shown that this pathogen causes mucosa-associated lymphoid tissue (MALT) lymphoma in both children and adults. In fact, when the organism is eradicated, extra gastric

**Table 3.** Endoscopic features associated to *Helicobacter pylori* infection: without anterior therapies versus with anterior

**anterior therapies, n (%)**

2 (11.76) 3 (25.0%)

*H. pylori* **infection with anterior** 

http://dx.doi.org/10.5772/intechopen.81437

**therapies, n (%)**

Endoscopical Aspects of *Helicobacter pylori* Gastritis in Children

The finding of *H. pylori-*associated gastritis without duodenal or gastric mucosal lesions puts the pediatric gastroenterologist in a dilemma on recommending eradication treatment. *H. pylori-*associated gastritis without PUD rarely gives rise of symptoms or progresses to severe complications of the disease during childhood [5]. The risk of *H. pylori*- associated cancer or MALT-lymphoma during childhood is extremely low in Europe and North America. Ohno reported two cases in Japanese children, a 14-year-old boy and another 6-year-old boy with MALT and *H. pylori* infection [5]. The lower risk of complications in children may be in part explained by a different immune response against the infection. In comparison with adults, gastric biopsies obtained from children infected with *H. pylori* show a lower degree of inflammation. In addition, a higher number of immunosuppressive regulatory T cells and a more prominent IL-10 mediated anti-inflammatory response have been detected in pediatric patients [1].

During childhood, *H. pylori* is associated with antral predominant gastritis and duodenal

In our study, the most frequent lesion identified by endoscopy was macroscopic antral nodular gastritis, which was present in 22 patients (78.86%). This high frequency is in accordance with a retrospective study from Japan that also found out a marked prevalence of nodular antral gastritis associated with *H. pylori* infection (98.5%) [7]. A nodular antral gastritis frequency of 82.53% was also reported by a Turkish study conducted in adults and adolescents


**Table 2.** Endoscopic features associated to *Helicobacter pylori* infection.

2 (16.67%) with antral hyperemia and 3 (25%) showed a normal mucosal aspect. In patients who were not previously treated, we observed macroscopic nodular antral gastritis in 15 cases (88.24%), antral hyperemia in 2 cases (11.76%) while a normal appearance of gastric mucosa was never detected (**Table 3**). There was not a statistically significant association between the severity of mucosal damage at endoscopy and the existence of a previous therapy against the infection (p = 0.06).

In our study, bleeding was the presenting symptom in 4 children; three of them had pan gastritis, and one had nodular gastritis and esophagitis.


**Table 3.** Endoscopic features associated to *Helicobacter pylori* infection: without anterior therapies versus with anterior therapies.
