Despite the fact that infection with Helicobacter pylori is still one of the most common bacterial infections all over the world, only a minority of infected individuals develop H pylori–related diseases such as gastric or duodenal ulcer, gastric adenocarcinoma, and non-Hodgkin gastric lymphoma (1). H pylori–infected individuals have a 6-fold increased risk of developing gastric cancer (2). The factors determining which H pylori–infected individuals develop a severe disease of the digestive tract remain unknown. The severity of gastric mucosa pathology in the stomach depends on the host immune response, host genetics, and bacterial genotype (3–7). H pylori shows a high degree of genetic variability. Genetic differences among bacteria infecting a human population probably play the main role in the clinical outcome of the infection. According to most studies in adults, the severity of H pylori–related disease correlates with the presence of cagA, vacA s1m1, and iceA1 genotypes (8,9). There have also been studies that did not confirm an association between specific genotypes and clinical outcome (10). The virulence role of other proposed H pylori genes, such as cagE and babA, has not yet been confirmed (11,12). Although the role of the cagA gene in the pathogenicity of H pylori has been confirmed in adults as well as in a pediatric population (13–15), there have been only a few and conflicting reports about the importance of vacA s1m1(16,17) and iceA1(18,19) for the degree of gastric mucosal damage in children. Furthermore, a distinct geographic distribution of the virulence genes has also been demonstrated (20,21). Data concerning virulence genotypes and genetic variations among different geographic regions in the pediatric population are still limited (13–19,22–33). To the best of our knowledge, 19 studies have so far been published in pediatric populations concerning the prevalence of H pylori genotypes and a possible association between polymerase chain reaction (PCR)–detected genotypes and gastric mucosal damage (reviewed in Table 1).
The aims of this study were to determine the prevalence of the genes cagA, vacA, and iceA in children from southeastern Europe and to correlate their presence with the severity of histological changes in the stomach, classified according to the Sydney scoring system (34). To the best of our knowledge, this prospective study includes the largest number of H pylori isolates from a single symptomatic pediatric population studied to date and is the first such study performed on children of southeastern European origin.
MATERIALS AND METHODS
Patients, Biopsies, Rapid Urease Test, and Histology
This prospective study included 165 consecutive children who presented with recurrent abdominal pain to the University Medical Center Ljubljana, Slovenia, from February 1999 to November 2005. All of the patients included in the study underwent upper gastrointestinal endoscopy performed by a pediatric gastroenterologist. Endoscopic diagnoses in the stomach were defined as follows: normal gross appearance, hyperemia, nodularity, erosions, and ulcers. At least 4 biopsy specimens were obtained from patients included in our study: 2 from the gastric antrum and 2 from the corpus of the stomach. One corpus and 1 antrum specimen were used for the rapid urease test H pylori Quick Test (Biohit Diagnostics, Helsinki, Finland). If the test was positive, then the child was included in the study. We also used samples from the rapid urease test for DNA extraction. The other 2 specimens were used for histopathological examination. Gastric biopsies were fixed in 4% formaldehyde solution phosphate buffered at pH 7.4, processed according to the standardized histochemical procedure and embedded in paraffin wax. Consecutive 4-μm sections were stained with hematoxylin and eosin. The following histological features were evaluated in each slide by a single experienced pathologist according to the updated Sydney classification (34): density of H pylori colonization (0–3), acute inflammation (0–3), chronic inflammation (0–3), atrophy (0–3), intestinal metaplasia (0–3), and surface epithelial damage (eg, erosion).
Patients who had received antibiotics, nonsteroidal anti-inflammatory drugs, proton pump inhibitors, and H2-receptor blockers in the last 30 days before the endoscope procedure were excluded from the study.
DNA Extraction and Genotyping of H pylori
For genotyping of H pylori, DNA was isolated from biopsy specimens previously used for the rapid urease test. Biopsy specimens were carefully removed from the transport medium to a 1.5-mL microcentrifuge tube using a sterile needle. After adding 1.0 mL of phosphate-buffered saline (PBS; 50 mmol/L potassium phosphate, 150 mmol/L NaCl, pH 7.2) and short vortexing, biopsy specimens were centrifuged at 8000 rpm for 1 minute. After removal of PBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and tissue protocol, following the manufacturer's instructions. The quality of isolated DNA and absence of PCR inhibitors were tested in all DNA samples by amplification of a 77-bp fragment of the H pylori ureA gene by real-time PCR, as previously described (35). The presence of the cagA gene was confirmed and identification of vacA genotypes (s1a/s1b/s1c, s2, and m1/m2/m2a/m2b) was performed using commercial reverse-hybridization assay RHA kit H pylori virulence (Labo Bio-medical Products, Rijswijk, the Netherlands), following the manufacturer's instructions, as described previously (36). The iceA genotypes were determined using a FastStart Taq Polymerase kit (Roche Diagnostics, Mannheim, Germany) and primers targeting the 247-bp fragment of iceA1 and 229 or 334-bp fragments of iceA2, as described previously (8,20).
The SPSS 11.0 statistical package (SPSS Inc, Chicago, IL) was used for data management and analysis. A P value of < 0.05 was considered statistically significant.
Informed consent was obtained from the parents of all of the patients and the protocol was approved by the state ethics committee.
A prospective analysis was performed on 165 children who had undergone gastroduodenoscopy and had been diagnosed with H pylori infection. There were 58 boys and 107 girls (age range 4–18 years, mean 13 years). The patient population was homogeneous and of southeastern European origin, except for 1 girl, whose parents originated from India. Most of the patients (140/165 and 84.8%) had recurrent abdominal pain as a leading referral symptom. In the other cases, the predominant health problems were vomiting (13/165, 7.8%), diarrhea (5/165, 3%), retrosternal pain (2/165, 1.2%), anemia (2/165, 1.2%), melena (1/165, 0.6%), regurgitation (1/165, 0.6%), and weight loss (1/165, 0.6%). All of the patients had endoscopic signs of antral gastritis. During the endoscopic procedure, we detected 11 children with erosion in the upper part of the gastrointestinal system: gastric erosion (9 of 11) and bulbar erosion (2 of 11). We diagnosed 1 patient with a gastric ulcer and 1 patient with a duodenal ulcer. The number of infected children in our study group with erosion and especially ulcers was small. One reason may be that the immune response to H pylori infection and also the genetics of Slovenian children do not favor development of severe damage to gastrointestinal mucosa. It is hard to believe that selection bias is the reason because the criteria for performing an endoscopy in children are the same as in other European countries.
A total of 144 specimens of gastric mucosa from 165 patients were available for histological analysis. The results are summarized in Table 2.
Prevalence of cagA, vacA, and iceA Alleles
All 165 samples included in the study were positive for the ureA gene, suggesting that DNA was successfully isolated and there were no apparent PCR inhibitors. The frequencies of cagA, vacA, and iceA alleles among the children included in our study group are shown in Fig. 1, from which it is clear that the cagA gene was found in 101 of 165 patients (61.2%).
The s-region and m-region of the vacA gene were detected in 150 of 165 isolates. The s1m1 genotype was the most frequent (63/150, 42%), followed by s1m2 (42/150, 28%) and s2m2 (36/150, 24%). The most frequent subtype was s1a (92/98), followed by s1b (6/98). We were unable to detect a subtype in 7 of 105 s1-positive samples. Genotype vacA s2m1 and subtype s1c were not detected in any of our biopsy samples. In 9 of 150 (6%) isolates, more than 1 vacA genotype was detected. Among iceA genes, the iceA1 allele predominated and was confirmed in 100 of 162 (62%) isolates. The iceA2 gene was detected in 50 of 162 (31%) strains. Both genes were contained in 12 of 162 (7%) biopsy samples. The iceA gene was not detected in 3 samples.
Correlation of cagA, vacA, and iceA Alleles With Histological Parameters in the Stomach
The associations between genotypes and histological parameters are shown in Tables 3 to 5. A strong correlation was demonstrated between cagA-positive status and H pylori density score (P < 0.01), activity of inflammation (P = 0.01), and degree of mononuclear cell infiltration (P < 0.01). No association was found between iceA1 expression and any of the histopathological parameters in the stomach (density score, degree of polymorphonuclear cell infiltration, degree of mononuclear cell infiltration [P = 0.35, P = 0.33, P = 0.46, respectively]). Although m1 and s1 alleles of the vacA gene were more common in specimens with a higher bacterial density score (P < 0.01, P < 0.01, respectively), no correlation was found between the m1 allele and acute or chronic inflammation (P = 0.07 and P = 0.13, respectively). Interestingly, type s1 strains were associated with a higher degree of chronic inflammation (P = 0.03), but not with the degree of polymorphonuclear cell infiltration (P = 0.26).
Intestinal metaplasia was observed in 3 patients and was accompanied by atrophy in 2 cases. We detected atrophy in 38.4% (48/125) children with nonmixed infection included in our study. There was no statistically confirmed association between cagA status and gastric atrophy (P = 0.87) or vacA s1 and gastric atrophy (P = 0.55). In addition, there was no correlation between iceA1-positive samples and the presence of mucosal atrophy (P = 0.48).
Correlation Between Genotypes and Intensity of Clinical Complaints
The duration of gastrointestinal complaints before the endoscopic procedure ranged from 0.5 to 60 months (mean duration 8.2 months). CagA-positive children came to the outpatient clinic 4 months later than the rest of the study population (P = 0.09). No significant association between the iceA1 allele and duration of abdominal pains was noted (P = 0.83). Although the vacA s1-positive patients were examined 5 months later than children with vacA s2 strains, the difference was not statistically significant (P = 0.06).
Infection With Multiple Strains
Mixed H pylori infections, defined by more than 1 vacA or iceA allele in the same biopsy sample, were detected in 19 (11.5%) of the 165 children studied. No association was found between multiple infections and the level of activity (P = 0.13) or chronicity of gastritis (P = 0.15). However, there was a correlation between the presence of multiple H pylori strains and the H pylori density score (P = 0.03). Patients with mixed H pylori infection were excluded from further analysis for genotype phenotype correlation.
We investigated the prevalence of cagA, vacA, and iceA genes and their relation to the degree of gastric mucosa pathology in 165 children from Slovenia infected with H pylori. To the best of our knowledge, this study included the largest number of children studied to date and is also the first pediatric study to investigate the importance of H pylori genes in children originating from southeastern Europe.
As shown in Table 1, the prevalence of the cagA gene among European children varies from 22.4% in Portugal (30) to 76% in Finland (23). In our study, 61.1% of children from Slovenia were positive for the cagA gene. CagA-positive strains are considered more virulent in most of the studies performed on adults (8,13). The cagA gene has at least a dual role. It is a marker for a pathogenicity island (PAI) and encodes the CagA protein (37). PAI encodes approximately 30 genes, such as the genes responsible for the type IV bacterial secretion apparatus and genes (cagG, cagH, and cagI) associated with a particular host cell response (37,38). The type IV secretion system is used for transferring protein CagA into the epithelial target cells. The result of phosphorylation of protein CagA is rearrangement of the host cell cytoskeleton (“hummingbird phenotype”) (37,39). In addition, it has been shown that the level of cytoskeletal changes is related to the number of tyrosine phosphorylation motifs located in the 3′ region of the cagA gene (40,41). As shown in Table 1, 11 pediatric studies have been published so far that have examined the association between cagA-positive status detected by genetic tests and gastric mucosal pathology. Interestingly, some authors (14,15,18,25,26) have described a significant correlation between the severity of histological changes and the presence of the cagA gene in the H pylori genome, whereas others (17,19,22,24,29,33) have been unable to confirm this association. A strong correlation was found in our study between cagA–positive status and the density of H pylori colonization, as well as the severity of acute and chronic inflammation.
Unlike cagA, the vacA gene is present in all strains of H pylori. The strains vary in their ability to produce cytotoxin according to the presence of different vacA s and m subtypes (42). Strains containing m1 and s1 are the most active in producing toxin, which causes vacuolation of the epithelial cells. Among s1 subtypes, s1a is more potent than s1b or s1c(43). Various studies have confirmed a specific geographic distribution for vacA alleles (21). For example, s1c is the major strain in east Asia, whereas s1a is the predominant strain in northern Europe and s1b in Portugal and Spain (8). In our population, the vacA s1m1 genotype was predominant and a large majority (92 of 98 specimens) was of the s1a subtype. Such a result was expected and is in agreement with other studies, especially because there is small diversity of ethnic backgrounds in Slovenia. When we subtyped the vacA gene, we found a correlation between vacA s1 alleles and the degree of bacterial density, as well as of chronic inflammation. These results are consistent with the study published by Singh et al (32). They subtyped the vacA gene from 52 infected children and were able to confirm the same correlation between the bacterial load and mononuclear cell infiltration and the vacA s1 genotype. In addition, gastric epithelial cell apoptosis was also higher in s1-positive children. Gusmao et al (16) confirmed the association between vacA s1 strains and peptic ulcer in children.
Recently, an additional H pylori gene, iceA, has been detected through its upregulation after adherence of the bacteria to gastric target cells. Gene iceA has 2 allelic variants: iceA1 and iceA2(44). As shown in Table 1, the prevalence of the iceA gene in a pediatric population was determined in 5 studies. The only European study, from Portugal, showed a clear predominance of the iceA2 genotype. IceA alleles were determined in 37 out of 119 children included in the study and 7.8% of those samples contained the iceA1 gene. These data may be biased because of the low number of patients. In other, non-European studies, the iceA1 genotype has been more frequently observed: 14% of iceA1-positive samples were obtained in Brazil (18), 37% in Israel (19), and 44% in North America (24). In contrast with other reports, iceA1 was found to be the most frequent genotype detected in our population, being present in 62% of the tested samples. Although it has been suggested that the iceA1 allele is associated with ulcer disease in adults (8), the degree of histological changes did not correlate in our study with subtype iceA1, confirming the results of other pediatric studies (19,24). Our results suggest that, in contrast with cagA and vacA s1 genotypes, the iceA1 allele is probably not associated with the degree of gastric inflammation in children infected with H pylori.
These data suggest that the prevalence of genes cagA, vacA s1m1, and iceA1 is high in southeastern Europe. According to the data from previous prevalence studies, it can be estimated that the frequency of the probably more virulent cagA and vacA s1 genes increases from the south to the north of Europe. This may also be the result of adaptation of H pylori to the environment (eg, climatic conditions, food) in different geographic regions.
On the basis of vacA and iceA genotyping, simultaneous infections with 2 or more H pylori strains were detected in 11.5% of our patients. Although the density of H pylori colonization was higher in these patients, the degree of gastric mucosa damage was similar to the group of patients infected by a single H pylori strain. This is in contrast with adult populations, in which multiple strain infection has been associated with a higher degree of gastric inflammation (22,45). It may be speculated that the different H pylori strains need time to act synergistically and cause more damage in the gastric mucosa than infection with a single strain.
Atrophic gastritis and intestinal metaplasia are considered to be precancerous lesions (46). It has been confirmed that long-lasting H pylori gastritis in adults causes the development of atrophic gastritis and intestinal metaplasia (47). The association between H pylori infection, atrophy, and intestinal metaplasia is not so well defined in pediatric populations (48,49). Our study demonstrated that the development of atrophic gastritis in children infected with H pylori is not dependent on infection with more virulent strains, such as cagA- or vacA s1-positive H pylori. These data suggest that other pathogenetic factors, such as the duration of infection, environmental factors, or other virulence genes, may trigger the development of gastric atrophy and intestinal metaplasia.
Finally, no association was observed between cagA, vacA s1m1, or iceA1 status and the frequency or degree of gastrointestinal complaints. Surprisingly, vacA s1- and cagA-positive children attended the doctor on average 5 months later. Korzon et al (14) correlated only the cagA genotype with the intensity of complaints and our observations are in agreement with the results of their study. Because we have no logical explanation for such results, additional studies are necessary to find the reason.
In conclusion, in a prospective study including the largest number of pediatric H pylori isolates studied to date, cagA, vacA s1a m1, and iceA1 genotypes were identified as the predominant genotypes of H pylori in southeastern European children. Our study confirms that the prevalence of genotypes cagA and vacA s1 decreases from northern to southern parts of Europe. Although cagA and vacA s1 genotypes are associated with higher H pylori density and more pronounced chronic gastritis, the iceA gene is probably not important for the virulence of H pylori in children. In addition, in childhood, cagA and vacA s1 may not be important in the development of gastric atrophy and intestinal metaplasia.
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