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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2012 Jul 18;93(4):295–304. doi: 10.1111/j.1365-2613.2012.00831.x

Correlation of serum antibody titres with invasive methods for rapid detection of Helicobacter pylori infections in symptomatic children

Khaled Abdulqawi *, Abeer M El-Mahalaway †, Amer Abdelhameed ‡, Alsayed A Abdelwahab §
PMCID: PMC3444986  PMID: 22804766

Abstract

Helicobacter pylori (H. pylori) is causally associated with peptic ulcer disease and gastric carcinoma. Typically, children get infected during the first decade of life, but diseases associated with H. pylori are seen mainly in adults. Multiple diagnostic methods are available for the detection of H. pylori infection. The aim of this study was to evaluate the correlation and diagnostic accuracy of three invasive methods [rapid urease test (RUT), histology and bacterial culture] and one non-invasive method (IgG serology) for diagnosis of H. pylori infection in a prospective cohort study conducted on 50 symptomatic children between two and eighteen years of age. Endoscopies with gastric biopsies were performed for RUT, culture and histopathological examination, respectively. IgG antibodies were measured in patient sera using a commercially available enzyme-linked immunosorbent assay (ELISA). RUT and positive H. pylori IgG antibodies were concordant in 88% (44/50) of patients. Both tests were negative in 32% (16/50), and both were positive in 56% (28/50). Disagreement occurred in 12% (6/50) of the patients: three of them (6%) had positive RUT and negative H. pylori IgG, and another three (6%) had negative RUT and positive H. pylori IgG. A combination of RUT with non-invasive serology constituted the optimum approach to the diagnosis of H. pylori infection in symptomatic children. The non-invasive serological test (ELISA) could not be used alone as the gold standard because it cannot distinguish between active and recently treated infection; and bacterial culture could not be used alone because of its low sensitivity.

Keywords: children, Helicobacter pylori, invasive, non-invasive

Helicobacter pylori (H. pylori) infection is almost always acquired in early childhood and usually persists throughout the patient's life unless a specific treatment is given (Rothenbacher et al. 2000). Helicobacter pylori infection is common in both developed (30–50% of adult) and developing countries (80%; Frenck & Clemens 2003).

H. pylori are important human pathogens, responsible for most peptic ulcer diseases, gastritis, gastric adenocarcinomas and gastric mucosa-associated lymphoid lymphoma, even in the pathogenesis of some extra gastric diseases. In most people, H. pylori infection is largely restricted to the gastric antrum (Chuan et al. 2005a).

The vast majority of gastrointestinal (GI) causes of iron deficiency anaemia (IDA) affect the upper GI tract, and, in particular, there is a high prevalence of conditions associated with iron malabsorption such as H. pylori-related pan gastritis (Vannella et al. 2008).

H. pylori are spiral-shaped gram-negative bacterium, about 3 µm long with a diameter of about 0.5 µm and with four to six lophotrichous flagella. Helicobacter pylori is microaerophilic with optimal growth at O2 levels of 2–5% with an additional need for 5–10% CO2 and high humidity (Kusters et al. 2006).

The powerful enzyme urease is used by H. pylori to maintain a favourable pH by breaking down the urea in the stomach into ammonia and bicarbonate–strong bases that counteract the acid in the stomach (Chalmers et al. 2004). Assessment of urease activity is the basis for the detection of H. pylori by a rapid urease test (RUT; Versalovic 2003).

Both invasive and non-invasive tests are used for diagnosis of H. pylori. Invasive tests include rapid urease, histology and bacterial culture, which required endoscopy to obtain biopsies of the gastric mucosa. Non-invasive tests include the detection of the bacterial urease by the 13C-, or 14C-urea breath tests are best, but are more expensive, not always available (poor feasibility limit), difficult to apply to non-compliant children, and have an uncertain cut-off level in paediatric patients remains unsettled (Tamara et al. 2005). Serology to detect IgG and IgA responses to the organism have also become available (Redéen et al. 2011) and a rapid enzyme immune assay (EIA) to detect H. pylori antigen in stool has been developed (Rajindrajith et al. 2009). In adults, the stool test has a sensitivity and specificity of 90%. However, it has the disadvantage that stool samples are difficult to collect and handle (Gisbert & Pajares 2004). Polymerase chain reaction (PCR) is also possible to detect stool H. pylori but is of limited value because of the presence of inhibitors of H. pylori DNA amplification in faeces. Also, H. pylori is difficult to culture from stool (Kathleen et al. 2004). At present, there is no single test for H. pylori that can be used as the gold standard (Robert et al. 2006), and the choice of a specific test for patient depends on local experience and the clinical setting (Rajindrajith et al. 2009).

Thus the present study aimed to evaluate the correlation and diagnostic accuracy of three invasive methods (RUT, histology and bacterial culture) and one non-invasive method [IgG serology as judged by an enzyme - linked immunosorbent assay test (ELISA)] for H. pylori infection diagnosis in children with dyspeptic symptoms.

Patients and methods

Fifty children with dyspeptic symptoms (recurrent abdominal pain, vomiting, diarrhoea, anorexia, failure to thrive, iron deficiency anaemia) selected from the Pediatric and Tropical Medicine Departments, Al-Jedaany Hospitals, Jeddah, KSA, were enrolled in this study in the period from November 2007 to April 2011. The age of the children ranged from 2 to 18 years. Patients were excluded from the study who had taken antimicrobial agents, antacids, H2 blockers, a proton-pump inhibitor or bismuth subsalicylate within 4 weeks of the endoscopy, and they had been previously documented to be infected with H. pylori, had a known bleeding disorder or had a previous endoscopy.

Methods

The study was approved by the clinical research committee of the hospital and performed according to ethical procedures. A written informed consent was obtained from the patients' parents for the participation in our study. A structured questionnaire was given to the child's parents to collect information on the presenting symptoms. All the cases were subjected to full history taking, thorough clinical examination, and weight and height measurements.

For the detection of H. pylori infections, endoscopic gastric biopsies were obtained for RUT, histopathology and bacterial culture respectively, and a blood sample was taken for the anti H. pylori IgG antibody ELISA test.

As there is no single gold standard test for H. pylori, the operational gold standard definition of H. pylori infection was used. The definition was as follows: (i) Patients with positive culture results were considered infected. (ii) Patients with negative culture results were considered infected when both RUT and histology were found to be positive. (iii) Patients whose test result was found to be negative by all three gastric biopsy specimen-based tests were considered non-infected. (iv) Patients negative by culture and positive by either RUT or histology were considered indeterminate and when serological tests were positive, patients were considered infected (Sufi et al. 2008).

Gastric biopsy

Endoscopy was performed under general anaesthesia or conscious sedation [midazolam (0.2 mg/kg) and meperidine (1 mg/kg)]. Three antral biopsies were taken within about 2 cm of the pyloric channel using an Olympus (Center Valley, PA, USA) ZIF XQ230 fibre-optic endoscope; a RUT was performed on the first biopsy. The second biopsy was subdivided and was sent for histopathology (light microscopy, scanning electron microscopy and transmission electron microscopy studies). The third biopsy was sent for culture.

Blood collection

A 5-ml blood sample was collected within three days of endoscopy and before initiation of any therapy against H. pylori. After collection, the blood was kept at room temperature for 1 h, followed by centrifugation at 1500–2000 g for 10 min. The serum was aliquotted into cryovials and stored at −70 °C.

Rapid urease test

Immediately after collection one biopsy was tested for urease activity using the ‘hpfast’ test (GI Supply, Camp Hill, PA, USA) according to the manufacturer's instructions. In brief, the biopsy was placed in an agar gel containing urea and two pH dye indicators: bromthymol blue and methyl red. A positive test was interpreted with the change of the agar colour from yellow to dark green or blue within 24 h.

Histopathological evaluation

The histopathologist interpreed all biopsies unaware of the clinical status of the child. The classification and grading of gastritis was conducted according to the updated Sydney system (Dixon et al. 1996) which records the presence of inflammation (mononuclear cell infiltration), activity (neutrophil infiltration), atrophy and intestinal metaplasia, and preneoplastic changes.

Light microscopic studies

Tissue specimens were fixed in 10% neutral buffered formalin embedded in paraffin and cut in sequential 4–5-μm sections. These sections were stained with Giemsa (Gray et al. 1986) for the detection of H. pylori, hematoxylin and eosin (H&E; Lamberg and Rothstein 1979) for histopathological examination of gastric mucosa and Genta (Genta et al. 1994) for goblet cells.

Scanning electron microscopic study

Biopsy specimens were washed in saline, fixed in a mixture of 2.5% glutaraldehyde in (0.2 mol/l) cacodylate buffer (pH 7.4) for 24 h, then washed in two changes of cacodylate buffer, then post fixed for 2 h in osmium tetroxide (Hayat 1989). Specimens were dehydrated in ascending grade of ethyl alcohol and dried, and then fixed on scanning electron microscopy specimen holder, coated with gold and examined by JEOL Model JSM 35 scanning electron microscope, JEOL company, Tokyo, Japan.

Transmission electron microscopic study

Biopsy specimens were fixed in a mixture of 2.5% glutaraldehyde in (0.2 mol/l) cacodylate buffer (pH 7.4) for 24 h, then washed in two changes cacodylate buffer, then post fixed for 2 h in osmium tetroxide (Hayat 1989). The specimens were dehydrated in ascending grade of ethyl alcohol and embedded in Epon 812. The semithin section (1 μm) was cut by an ultramicrotome, stained with toluidine blue and examined by light microscope to show the tissue for good selection and localization of the needed part to examine in the thin section. The ultrathin sections (100 nm) were prepared and stained with uranyl acetate and lead citrate and examined by a JEOL model XC100 transmission electron microscope.

Bacterial culture

Immediately after collection the biopsy sample was placed in 0.1 ml sterile 0.85% saline. Half of the biopsy homogenate was placed on to a Columbia blood agar plate and the other half on to a Columbia blood agar plate with supplement (trimethoprim, vancomycin and polymyxin B) and streaked for isolation. The plates were incubated at 37 °C for up to 10 days and checked every alternate day for growth. On day 5 plates without obvious growth were sub-cultured onto a Columbia blood agar plate to promote the growth of lightly growing colonies that might be missed visually. All colonies suggestive of H. pylori were tested by Gram-stain, oxidase, catalase and urease tests to confirm the identification (Megraud 1997).

Serum anti-H. pylori antibodies ELISA testing

Serum samples were tested for the presence of anti-H. pylori IgG antibodies to specific H. pylori antigen (high molecular weight cell–associated protein (HM-CAP) using the HM-CAP ELISA kit (EZ-EM Inc, Westbury, NY, USA) according to the manufacturer's directions. Samples with an ELISA value of <1.8 ELISA units were considered negative, and samples with an ELISA value of >2.2 ELISA units were considered positive. Samples with values between 1.8 and 2.2 ELISA units were considered indeterminate and were retested. A sample that was still indeterminate after the repeat test was recorded as negative (Robert et al. 2006).

Analytical plan

Descriptive statistical tests were expressed as mean ± standard deviation. The differences between the groups were evaluated using the nonparametric Mann–Whitney U-test with the threshold of significance set at P < 0.05. Cut-off values of >2.2 U/ml H. pylori IgG were used, as these gave the best sensitivity, specificity, positive and negative predictive value and likelihood ratio.

Results

The study was carried out on 50 children with dyspeptic symptoms [36 males (72%) and 14 females (28%) whose ages ranged between two and eighteen years]. The demographic and clinical characteristics of the cohort patients are summarized in Table 1 and show that there were no statistical differences in demographic and clinical characteristics at time of admission of under 6-year-old age and over 6-year-old age groups (P > 0.05). The ages of the 50 patients ranged from 3 year and 5 months to 18 year [mean 10 years and 6 month (SD ± 2 year and 8 month)], of whom 64% (32/50) were over 6 years old and 36% (18/50) under 6 years old; 72% (36/50) were males and 28% (14/50) were females. Recurrent abdominal pain was present in 96% (48/50), vomiting was present in 70% (35/50), hematemesis in 16% (8/50) and chronic diarrhoea was in 10% (5/50).

Table 1.

Demographic and clinical characteristics

Demographic characteristics All ages no (%) <6 years no (%) >6 years no (%) P-value
Age mean ± SD (years) 10.6 ± 2.8 50 (100) 32 (64) 18 (36) 0.5
Sex
 Male 36 (72) 12 (38) 20 (62) 0.2
 Female 14 (28) 6 (33) 12 (67) 0.4
Clinical characteristics
 Recurrent abdominal pain 48 (96) 17 (94) 31(97) 0.7
 Vomiting 35 (70) 12 (67) 23 (72) 0.3
 Hematemesis 8 (16) 3 (17) 5 (16) 0.6
 Chronic diarrhoea 5 (10) 3 (17) 2 (6) 0.3
 Others 17 (34) 6 (33) 11 (33) 0.3
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P > 0.05 (<6 years group vs. >6 years group).

In Table 2, of the 50 patients, 44 (88%) showed the same results with RUT and H. pylori IgG antibodies, of which 16 (32%) were negative and 28 (56%) were positive. Only 6 (12%) showed different results with the RUT and H. pylori IgG antibodies, of which 3 (6%) had positive RUT and negative H. pylori IgG antibodies tests, and the other three patients (6%) were negative for RUT and had positive H. pylori IgG antibodies tests. Of the 50 patients, 40 (80%) had the same results of culture and H. pylori IgG antibodies, of which 14 (28%) were negative and 26 (52%) were positive. Only 10 (20%) had different results of culture and H. pylori IgG antibodies, of which five (10%) were positive for culture and had negative H. pylori IgG antibodies tests, and the other five patients (10%) were negative for culture and had positive H. pylori IgG antibodies tests. Of the 50 patients, 38 (76%) had the same results of culture and RUT, of which 13 (26%) were negative and 25 (50%) were positive. Only 12 (24%) had different results of culture and RUT, of which seven (14%) were positive for culture and had negative RUT, and the other five patients (10%) were negative for culture and had positive RUT tests. The endoscopic findings in Table 3 were normal in 16 (32%) of 50 patients and abnormal in 34 (68%). Of the 16 patients with a normal endoscopic examination, 12 (75%) were negative for H. pylori, and 4 (25%) were positive for H. pylori. Of the 34 patients with an abnormal endoscopic examination, 28 (82%) were positive for H. pylori, and 6 (18%) were negative for H. pylori. Cases with abnormal endoscopy showed gastritis in 50% (17/34), esophagitis in 35% (12/34), duodenal ulcer in 18% (6/34) and duodenal erosions in 3% (1/34).

Table 2.

Helicobacter pylori IgG, rapid urease test (RUT) and culture among studied cases

Test Number (%)
Positive RUT and positive H. pylori IgG antibodies 28 (56)
Negative RUT and negative H. pylori IgG antibodies 16 (32)
Positive RUT and negative H. pylori IgG antibodies 3 (6)
Negative RUT and positive H. pylori IgG antibodies 3 (6)
Positive culture and positive H. pylori IgG antibodies 26 (52)
Negative culture and negative H. pylori IgG antibodies 14 (28)
Positive culture and negative H. pylori IgG antibodies 5 (10)
Negative culture and positive H. pylori IgG antibodies 5 (10)
Positive culture and positive RUT 25 (50)
Negative culture and negative RUT 13 (26)
Positive culture and negative RUT 7 (14)
Negative culture and positive RUT 5 (10)
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Table 3.

Endoscopic findings

Normal No (%) Abnormal No (%)
Total 16/50 (32) 34/50 (68)
H. pylori positive cases 4/16 (25) 28/34 (82)
H. pylori negative cases 12/16 (75) 6/34 (18)
Gastritis 0.0 17/34 (50)
Oesophagitis 0.0 12/34 (35)
Duodenal Ulcer 0.0 6/34 (18)
Duodenal erosions 0.0 1/34 (3)
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In Table 4, the findings show the sensitivity, specificity, positive predictive value, negative predictive value, the likelihood ratio for positive results (LR+), the likelihood ratio for negative results (LR−) and the accuracy of serum H. pylori antibodies levels for diagnosis of H. pylori infection in symptomatic children (cut-off values of >2.2 U/ml H. pylori IgG were used). The greatest sensitivity was achieved by the RUT (98%), followed by histology (92%), culture (90%) and lastly by serology (70%). The highest specificity was obtained by histology (100%) and culture (100%) followed by the RUT (89%) and serology (89%).

Table 4.

The performance characteristics of Helicobacter pylori IgG, rapid urease test (RUT), histology and culture

Test Sensitivity (CI 95%) Specificity (CI 95%) PPV (%) NPV (%) Likelihood ratio positive Likelihood ratio negative Accuracy (%)
H. pylori IgG 70 (54.2–80.3) 89 (73.4–96.1) 76 68 8.0 0.8 85
RUT 98 (90.1–99.2) 89 (70.4–89.8) 88 98 11.1 1.0 92
Histology 92 (81.4–96.2) 100 (92.0–100) 100 90 9.2 0.9 95
Culture 90 (78.6–94.4) 100 (92.0–100) 100 75 9.0 0.9 86
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PPV, positive predictive value; NPV, negative predictive value.

Histopathological findings

A section of stomach antrum in a H. pylori-infected patient showed a rod-shaped organism stained blue by Giemsa stain along the luminal surface and mucosa (Figure 1a). In Figure 1(b), chronic gastritis was evident histologically by the presence of inflammatory infiltrates of mononuclear cells: macrophages, lymphocytes and plasma cells). There was a neutrophil infiltrate in the foveolar epithelium and superficial lamina propria and a lymph follicle with a germinal cap was present in the lower portion of the mucosa. Figure 1(c) showed heavy infiltration of the full thickness of the mucosa with chronic inflammatory cells (lymphocytes, and plasma cells). The gastric glands were compressed and separated by the inflammatory cells. In Figure 2(a), atrophy was recognized as judged by the loss of the normal gastric glandular cells. In Figure 2(b), metaplasia was recognized histologically by the presence of goblet cells, absorptive cells in the surface epithelium and glands and mild infiltration of the lamina propria by chronic inflammatory cells including lymphocytes, and plasma cells. Figure 2(c) shows an intestinal type of columnar epithelium with goblet cells which stain blue with alcian blue replacing gastric epithelial cells. There was a with mild infiltration of the lamina propria by chronic inflammatory cells.

Figure 1.

Figure 1

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A section of stomach antrum in a Helicobacter pylori-infected patient showing (1-a) rod-shaped organism (arrow) stained blue along the luminal surface and in the luminal mucosa (Giemsa ×1000). (1-b) Chronic inflammatory cells (C) infiltrate the foveolar epithelium and superficial lamina propria and lymph follicle with germinal cap (F) present in the lower portion of mucosa (H&E ×100). (1-c) Heavy infiltration of full thickness of mucosa with chronic inflammatory cells (C) and the glands (G) are compressed (H&E ×400).

Figure 2.

Figure 2

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A section of stomach antrum in a Helicobacter pylori-infected patient showing (2-a) atrophy of the gastric mucosa (arrow) (H&E ×200). (2-b) Intestinal type of columnar epithelium with goblet cells (arrow) replaced atrophic gastric mucosa and with mild infiltration of the lamina propria by chronic inflammatory cells (C) (H&E ×200). (2-c) Intestinal type of columnar epithelium with goblet cells (arrow) stained blue with alcian blue replaced atrophic gastric epithelial cells and glands and with mild infiltration of lamina propria by chronic inflammatory cells (C) (Genta ×200).

Scanning electron microscopy

Figure 3 shows a representative section of the gastric antrum from a H. pylori-infected patient showing pili (adhesive hair-like organelles that protrude from the surface of bacteria) and ruffle formation (intimate adherence of bacteria on to the gastric epithelial cells; Figure 3a). Figure 3(b) shows degeneration and necrosis of gastric epithelial cells in association with H. pylori infection.

Figure 3.

Figure 3

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Scanning electron micrograph of a section of stomach antrum in Helicobacter pylori-infected patient showing (3-a) pili and ruffle formation of Helicobacter pylori (arrow) on the gastric epithelial cells (×800). (3-b) Degeneration and necrosis of gastric epithelial cells (×600).

Transmission electron microscopic results

Figure 4 shows a representative section of gastric antrum from a H. pylori-infected patient. There are spiral bacteria adherent by filament-like structures to gastric epithelial cells that contain numerous electron dense secretory granules (Figure 4a). Figure 4(b) shows intestinal metaplastic columnar absorptive cells with surface microvilli and intracytoplasmic H. pylori.

Figure 4.

Figure 4

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Transmission electron micrograph of a section of stomach antrum in Helicobacter pylori-infected patient showing (4-a) gastric lumen (L) and spiral bacteria (S) adherent by filament-like structure (arrow) to gastric epithelial cells and its cytoplasm contain numerous electron dense secretory granules (E) (×20,000). (4-b) Intestinal metaplastic columnar absorptive cells with surface microvilli (M) and intracytoplasmic Helicobacter pylori (arrow) (×17,000).

Discussion

Current tests used to detect H. pylori are either invasive (rapid urease, histology and bacterial culture and PCR) or non-invasive (serological tests, 13C-urea breath test). These tests vary in their sensitivity and specificity, and the choice of test will depend on the situation, for example, whether the test is to detect infection or the success of eradication treatment (Mégraud 1996).

Each one has advantages and disadvantages. None of the methods can be considered the gold standard for the diagnosis of H. pylori infections. The tendency has been to use a combination of tests in both adult and paediatric studies (Lake & Chey 2001; Robert et al. 2006).

In our study, we evaluate the correlation and diagnostic accuracy of three invasive methods (RUT, histology and culture) and one non-invasive method (IgG antibodies serologic ELISA test) for the diagnosis of H. pylori infection in symptomatic children. Of the 50 patients, 44 (88%) showed the same results with RUT and H. pylori IgG antibodies, of which 16 (32%) were negative and 28 (56%) were positive. Only 6 (12%) showed different results between RUT and H. pylori IgG antibodies respectivley, of which 3 (6%) had positive RUT and negative H. pylori IgG antibodies tests, and the other three patients (6%) were negative for RUT and had positive H. pylori IgG antibodies tests. Thus of the 50 patients, 40 (80%) had the same results of culture and H. pylori IgG antibodies, and of these 14 (28%) were negative and 26 (52%) were positive. Only 10 (20%) had different results between culture and H. pylori IgG antibodies, of which 5 (10%) were positive for culture and had negative H. pylori IgG antibodies tests, and the other five patients (10%) were negative for culture and had positive H. pylori IgG antibodies tests. Of the 50 patients, 38 (76%) were with the same results of culture and RUT, of which 13 (26%) were negative and 25 (50%) were positive. Only 12 (24%) had different results between culture and RUT, of which 7 (14%) were positive for culture and had negative RUT, and the other five patients [10% (5/50)] were negative for culture and had positive RUT tests.

In spite of the difficulty in obtaining paediatric biopsies, conducting a study in a paediatric population and small cohort number, according to the updated Sydney classification the gold standard for H. pylori diagnosis remained histology on endoscopic samples (Dixon et al. 1996).

The standard H and E hematoxylin and eosin stain demonstrates the extent of inflammation (gastritis type) and H. pylori itself. Special stains were used to detect H. pylori if there are fewer present. Giemsa staining is the most widely used because it has good sensitivity, excellent specificity, and a lack of technical difficulty in preparation. Genta (H&E, Steiner silver, and Alcian blue combined) detects metaplastic epithelium easily because of its very characteristic goblet cells. The silver stain detects organisms in small numbers but its disadvantage is that it is not possible to study the histology properly with silver (Laine et al. 1997; Rajindrajith et al. 2009).

Our findings, also demonstrated by Hala (2006), showed that H. pylori are found throughout the stomach in the early stages of disease, and that H. pylori-associated inflammation was often mild, superficial or even absent in the gastric corpus.

H. pylori are gram-negative rods that have the ability to colonize and infect the stomach. The bacteria survive within the mucous layer that covers the gastric surface epithelium and the upper portions of the gastric foveolae. The infection is usually acquired during childhood. Once the organism is acquired, is passed through the mucous layer and is established at the luminal surface of the stomach, an intense inflammatory response of the underlying tissue develops. This is then associated with tissue damage and the histological finding of both an active and chronic gastritis (Raúl et al. 2006).

The host response to H. pylori and bacterial products is composed of T- and B-cell lymphocytes, denoting chronic gastritis, followed by the infiltration of the lamina propria and gastric epithelium by polymorphonuclear leucocytes that eventually phagocytize the bacteria. Gastric epithelial cells release proinflammatory cytokine interleukin (IL)-8 and express class II molecules, which increase inflammation (Nogueira et al. 2001; Rugge & Genta 2005).

Johanna et al. (2003) reported that lymphocytic gastritis was commonly associated with H. pylori infection. Ricuarte et al. (2005) reported that the presence of a higher density of mucosal mononuclear cells was a predictor for the presence of gastric atrophy in Colombian children with atrophy, suggesting that a high density of mononuclear cells that infiltrate deep into the lamina propria may be a precursor to atrophy. Our findings also demonstrated by Hala (2006) reported that inflammation progresses from the antrum into the adjacent corpus and leads to a reduction in acid secretion and eventually loss of parietal cells and development of atrophy. Chuan et al. (2005b) reported that H. pylori infection may provide the proper environment for the occurrence of atrophic gastritis and intestinal metaplasia. George and Frederic (2006 reported that gastric atrophy and intestinal metaplasia exist in children and sometimes in young children.

Examination of tissue sections by scanning electron microscopy demonstrated degeneration and necrosis of gastric epithelium because of adherence of H. pylori to epithelium and ruffle formation. Many previous reports have described the ability of H. pylori to adhere to epithelial cells by interaction of bacteria with mucosal cell receptors and cytotoxin release by H. pylori causing severe damage to gastric epithelium (Bianca & Thomas 2011). Our findings were similar to those reported by, Kwok et al. (2002) and Shaffer et al. (2011) who had also observed the presence of H. pylori pili and the ruffle formation on gastric epithelium.

Examination of tissue sections by transmission electron microscopy demonstrated spiral bacteria adherent to gastric epithelial cells by filament-like structure gastric epithelial cell cytoplasm containing numerous electron dense secretory granules. Adhesion was considered an important aspect of bacterial pathogenicity and defined as a close attachment between bacteria and epithelial cells, such that virtually no space was visible between them using transmission electron microscopy. Adherence affords an advantage for toxin-producing organisms and induces degeneration of microvilli, degeneration of the cytoskeleton with actin polymerization, depletion of mucus granules and an increase in sialic acid-rich glycoproteins in the apical part of the cytoplasm (Poutahidis et al. 2001; Dubois & Borén 2007). In this study, H. pylori were observed in the cytoplasm of metaplastic columnar absorptive cells. Previous studies using transmission electron microscopy demonstrated the morphologic features of H. pylori and its ability to invade epithelial cells. The organism has invasive potential and can be demonstrated in the epithelial cells and intercellular tight junctions (Nirag et al. 2003). Thus our electron microscopic findings confirmed the histopathological findings at the cellular level.

Our study demonstrated that the RUT has high sensitivity (98%) and low specificity (89%) in diagnosis of H. pylori. The low specificity is because of false-positive results. These findings are in accordance with the previous studies (Silvio et al. 2001; Robert et al. 2006). In agreement with our findings, Roma-Giannikou et al. (2010) reported a low sensitivity of RUT in children, This may be related to the low H. pylori load in biopsy samples from children, and the need to use a full sample rather than a split sample as is often used in adults. The association of RUT and histology produces better accuracy in for confirming the diagnosis of infection H. pylori because of the high sensitivity of RUT and the high specificity of histology methods. These findings are in accordance with the previous studies (Kathleen et al. 2004; Mahmood and Hamid 2010).

In this study, the bacterial culture had low sensitivity (90%) in comparison with what has been reported in other studies (Destura et al. 2004; Falsafi et al. 2007). However, its specificity was high (100%) providing incontestable proof of the presence of bacteria (Chey & Wong 2007; Mégraud & Lehours 2007). The disadvantages of biopsy-based methods are risk of anaesthesia, patient discomfort and inconvenience, high expense, availability limited to specialized centres and sampling error because of the patchy nature of the infection and low concentration of bacteria in fragments. Difficulties in isolation and culturing, technical requirements and relatively low availability made the bacterial culture method difficult to be used as the gold standard (Mégraud 1996).

The sensitivity and specificity results of H. pylori IgG antibodies by ELISA assay test in children were 70% and 89% respectively. A lower sensitivity for serological H. pylori tests in children compared with adults has been reported (Angelika et al. 2001; Robert et al. 2006). Other studies reported a higher sensitivity for serological H. pylori tests in children (Obata et al. 2003; She et al. 2009) and a higher specificity (Thomas & De Sousa 2006; Abd El-Latif et al. 2011).

Serological tests are commercially available, easy to perform and inexpensive and therefore have been recommended for the diagnosis of H. pylori infections in adults (Tanih et al. 2008; Veijola et al. 2010). Many serological tests, mainly IgG based, have been validated in adult populations against invasive methods with acceptable sensitivity and specificity for clinical use (Megraud 1997; Salomaa-Räsänen et al. 2004; Abusini et al. 2009). Serological studies in children showed controversial results, with a wide range of sensitivity (50–96%) and specificity (83–100%; Angelika et al. 2001) Both the sensitivity and the specificity of serological test kits depend on the antigen preparation used (Janulaityte-Gunther et al. 2005). Different immune responses of the populations under investigation may explain the differences in sensitivity of the same test in children of different ages or geographical origins (Leal et al. 2008). There are differences in the antigenicity of the multiple H. pylori strains and even of the different antigens of the same strain. Other than the differential immune responses at different ages, duration of infection may play a role. Because the primary infection occurs most commonly in infants and toddlers, younger children are expected to have a shorter duration of infection than older children and adults (Mohammad et al. 2008).

The systemic immune response against H. pylori typically shows a transient rise in specific IgM antibodies, followed by a rise in IgG and IgA antibodies that persist during infection. Serum IgM and IgA antibodies tests against H. pylori are transiently detected and unreliable in detecting gastric colonization. They have little value for the serological diagnosis of infection (Leal et al. 2008), and therefore, only IgG antibodies are used in clinical laboratory practice, which is measured using ELISA. The presence of IgG antibodies against H. pylori denotes active infection because once an individual is colonized, the infection continues throughout life unless a course of appropriate eradication therapy is instituted (Rajindrajith et al. 2009).

Antibody-based tests had developed during the last decades. These tests differ in a number of features: antigen composition (e.g. different H. pylori strains), antigen source (e.g. native or recombinant), protocols for antigen purification, class of immunoglobulin detected (e.g. IgG, IgA, IgM), origin of samples (e.g. serum, saliva, urine) and test source (i.e. commercial and in-house test). The main advantages of antibody-based tests are their simplicity, low cost, speed and minimal patient discomfort (Leal et al. 2008).

Serological tests for H. pylori infection have been helpful in epidemiological studies of prevalence, mode of transmission and spontaneous clearance of the infection; allowing for the development of preventive measures of infection early in life and confirming the presence of H. pylori infection in case of equivocal results of the other diagnostic methods (Paula & Jaques 2004; Leal et al. 2008).

However, serological assays are unable to distinguish between the current and past infection, are not appropriate for monitoring the treatment response because of the slow decrease in antibody titre after cure and do not give additional information about inflammation or mucosal damage (Brendan et al. 2000; Sultan & Cuffari 2010).

Age and specimen handling may explain the low sensitivity in antibody detection by ELISA. Leal et al. (2008) meta-analysis included children with a wide range of ages (< 1–19 years) mentioned that, the ability to mount an efficient immune response varies with age, showing a weaker response during the first years of life. Concerning specimen handling, the majority of studies used frozen sera: thus, samples from different studies may have been subjected to diverse freeze–thaw cycle histories that may affect sensitivity. ELISA tests in children showed a high specificity and positive likelihood ratio, but low sensitivity and high negative likelihood ratio (Sultan & Cuffari 2010).

Patient compliance with H. pylori eradication therapy may be better after endoscopy, compared with diagnosis of the infection by non-invasive testing. Better compliance results in a lower rate of treatment failures and therefore a reduced risk of therapy-induced antibiotic resistance of the surviving bacteria (Brendan et al. 2000).

We demonstrated that the combination of more than one method to determine H. pylori infection provides enhanced sensitivity and specificity to one method alone. Our findings suggested that the association of RUT and histology together produces a better accuracy for confirming diagnosis of infection. However, based on our results, we agree with the revised concluding statement that the RUT in addition to both histology and serology would be the best choice. We feel it is a valid argument that the histology requires specialist knowledge, can differ between centres depending on the level of experience of the resident histopathologist and thus is not a universally consistent method. In addition, the selection of the disease tissue is subject to sampling error and dependent on the technical abilities of the endoscopist. Conversely, serological tests are easy to perform and can be universally applied.

Conclusions

Non-invasive serological test (ELISA) cannot distinguish between the active and recently treated infection, so it cannot be used alone as the gold standard test, the low sensitivity of bacterial culture method means that it cannot be used as the gold standard. Association of the RUT and histology with non-invasive method of serology constitutes the best choice for the diagnosis of H. pylori infection in symptomatic children.

Acknowledgments

The authors would like to thank the Pediatrics and the Tropical medicine Departments staffs at Al-Jedaany hospitals, Jeddah, KSA for providing us an opportunity to perform our study.

Disclosure

There were no conflicts of interest.

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