Antonie van Leeuwenhoek (2014) 106:637–645 DOI 10.1007/s10482-014-0234-0

ORIGINAL PAPER

Prevalence of the Helicobacter pylori babA2 gene and correlation with the degree of gastritis in infected Slovenian children Matjazˇ Homan • Anja Sˇterbenc • Bosˇtjan J. Kocjan • Bosˇtjan Luzar • Nina Zidar Rok Orel • Mario Poljak



Received: 13 April 2014 / Accepted: 7 July 2014 / Published online: 24 July 2014 Ó Springer International Publishing Switzerland 2014

Abstract The aims of our study were to determine the prevalence of the babA2 gene within Helicobacter pylori strains circulating in the Slovenian pediatric population, to further clarify its significance in causing inflammation of gastric mucosa in children and to verify whether cagA, vacA, iceA and babA genes work independently or synergistically in causing gastritis. A total of 163 H. pylori isolates obtained from the same number of children were tested for the presence of cagA, vacA and iceA genes using previously established methods, while the babA2 gene was determined using novel polymerase chain reaction assay targeting a 139-bp fragment of the central region of babA2. The babA2 gene was detected in 47.9 % of H. pylori samples. The presence of the babA2 gene was strongly associated with cagA, vacA s1 and vacA m1 genotype. The babA2 status correlated positively with bacterial density score, activity of inflammation and chronic inflammation of gastric mucosa. No significant M. Homan (&)  R. Orel Department of Gastroenterology, Hepatology and Nutrition, University Children’s Hospital, Ljubljana, Slovenia e-mail: [email protected] A. Sˇterbenc  B. J. Kocjan  M. Poljak Faculty of Medicine, Institute of Microbiology and Immunology, University of Ljubljana, Ljubljana, Slovenia B. Luzar  N. Zidar Faculty of Medicine, Institute of Pathology, University of Ljubljana, Ljubljana, Slovenia

correlation was found between the babA2 status and the presence of atrophy or intestinal metaplasia. In addition, the activity of gastric inflammation and density score were significantly associated with the coexpression of the cagA, vacA s1, vacA m1 and babA2 genes. The study, which included the largest number of pediatric H. pylori samples to date, confirmed that babA2 gene plays an important role in the pathogenesis of H. pylori gastritis in children. Furthermore, our results suggest that babA2, cagA and vacA s1 and m1 gene products may work synergistically in worsening the inflammation of gastric mucosa. Keywords Helicobacter pylori  babA2 gene  Gastritis  Children

Introduction Infection with Helicobacter pylori is still the most common bacterial infection in humans, despite the significant decrease in its incidence recorded in recent years, especially in the developed world (Klindermann and Lopes 2009). Chronic inflammation of the gastric mucosa can cause peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma (Ertem 2013). In addition, the occurrence of extragastric diseases, such as idiopathic thrombocytopenic purpura and iron-deficiency anemia, are induced by H. pylori infection (Pellicano

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Table 1 Summary of babA2 prevalence studies in children and its association with the level of gastric or duodenal mucosal damage Country, reference*

No. of children

babA2 detection method

Frequency of babA2 positivity (%)

Clinical relevance of babA2

Portugala

58

Gerhard et al. (1999)

17.2

NA

USAb

61

Gerhard et al. (1999)

36.0

NA

USAc

47

Salama et al. (2000)

60.0

Not significant

Brazild

45

Gerhard et al. (1999)

84.4

NA

Bulgariae

16

Gerhard et al. (1999)

66.7

NA

Portugalf

117

Gerhard et al. (1999)

29.1

Significant

Turkeyg

49

Sheu et al. (2003)

40.8

Not significant

Slovenia, current study

163

novel assay

47.9

Significant

NA data not presented * Data were obtained from a(Oleastro et al. 2003), b(Podzorski et al. 2003), c(Talarico et al. 2009), d(Garcia et al. 2010), e (Boyanova et al. 2010), f(Oleastro et al. 2010), g(Ozbey et al. 2013)

et al. 2009). The frequency of clinical consequences among children infected with H. pylori is fairly low and only a minority of chronically infected children has symptoms because of H. pylori (Koletzko et al. 2006). Several bacterial, host and environmental factors have been studied in the last two decades to determine the clinical outcome of H. pylori infection (Yamaoka 2012; Delahay and Rugge 2012; Sgouros and Bergele 2006). Among bacterial factors, virulence genes are most important in defining the disease severity in infected patients (Yamaoka 2010; Suzuki et al. 2012). Previous studies performed in both adults and children

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have clearly shown that the severity of the disease depends mainly on the presence of cagA and vacA s1 alleles (Jang et al. 2010; Homan et al. 2009; Gwack et al. 2006). Recent data also suggest that genes that encode outer membrane proteins (OMPs), may also contribute to the pathogenicity of H. pylori. OMPs enable the adherence of H. pylori to the gastric mucosa and its permanent colonization. Several OMPs have been identified: SabA, AlpA, AlpB, OipA, HopZ and BabA (Ilver et al. 1998), of which BabA has been the most intensively studied. Four bab members of the paralogous family of OMPs have been described so far: babB, babC, babA1 and babA2 (Matteo et al. 2011). The babA2 gene encodes protein BabA, a blood-group antigen-binding adhesin, which mediates adherence of the bacteria to human Lewisb blood-group antigens on gastric epithelial cells. The babA1 gene is a ten-nucleotide sequence shorter than babA2 and cannot interact with blood-group antigen (Ilver et al. 1998). The babB and babC genes are babA’s closely related paralogues, with unknown function(s) (Colbeck et al. 2006). Most of the studies in the adult population have suggested a positive association between the presence of the H. pylori babA2 gene and the severity of inflammation of gastric mucosa (Olfat et al. 2005; Oleastro et al. 2003). In contrast, the pathogenic role of BabA protein in H. pylori-related gastritis in children remains unclear. To the best of our knowledge, seven studies dealing with H. pylori babA2 prevalence in children can be identified in peerreviewed journals (Oleastro et al. 2003; Podzorski et al. 2003; Talarico et al. 2009; Garcia et al. 2010; Boyanova et al. 2010; Oleastro et al. 2010; Ozbey et al. 2013) but with significantly different results, precluding any definite conclusion (Table 1). The aims of this study were (i) to determine the prevalence of the babA2 gene within H. pylori strains circulating in the Slovenian pediatric population and to further clarify its significance in causing gastric inflammation in children infected with H. pylori and (ii) to establish the relationship between different H. pylori babA2/cagA/vacA genotypes and the severity of histological changes in the stomach. To the best of our knowledge, this study included the largest number of H. pylori samples from children tested for the presence of babA2 to date.

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639

Materials and methods

Table 2 Sequences of the PCR primers designed for amplification of a 139-bp fragment of the babA2 gene

Study population

Primer

Sequence (50 –30 )

Nucleotide positionsa

babA (B)-F1

TATCAAGCCGTGCTTTT (s)

598–614

babA (B)-F2

TATCAGGCCGTGCTTTT (s)

598–614

babA (B)-F3

TATCAAGCGGTGCTTTT (s)

598–614

babA2krew2

CAACGAGCCAGGGTATC (as)

720–736

All H. pylori positive children that had undergone upper endoscopy in the main Slovenian pediatric hospital University Children’s Hospital in Ljubljana from 1999 until 2010 were included in the study. Patients who had received antibiotics, non-steroidal anti-inflammatory drugs, proton pump inhibitors and H2 receptors blockers in the last 30 days before the endoscopic procedure were excluded. A total of 223 eligible children were included: 165 children were from the period 1999–2005 and have previously been described in detail (Homan et al. 2009) and 58 children were newly diagnosed patients in the period 2006–2010. During endoscopy, at least two biopsies from the antrum and two from the corpus of the stomach were obtained. Two biopsy specimens were used for the rapid urease test using H. pylori Quick Test (Biohit Diagnostics, Helsinki, Finland). If tested positive, the patient was included in the study. The remaining specimens were used for histopathological examination. The gastric biopsies were prepared for histological analysis as described previously (Homan et al. 2009). The following histological features were evaluated and graded according to the updated Sydney classification, by two experienced pathologists: density of H. pylori colonization (0–3), activity of inflammation (0–3), chronic inflammation (0–3), atrophy (0–3), intestinal metaplasia (0–3) and surface epithelial damage (such as erosions) (Dixon et al. 1996). Each sample was evaluated blindly by two pathologists to ensure consistency. Any discordant evaluations were additionally reviewed to achieve a consensus. Study protocol DNA extraction was performed from the biopsy samples used for the rapid urease test, as described previously (11). Following DNA extraction, samples were quantified using a NanoDrop ND-2000c spectrophotometer (Nanodrop Technologies, Oxfordshire, UK) and stored at -20 °C until use. The cagA gene was identified and vacA and iceA genotypes determined, as described previously (Homan et al. 2009).

s Sense, as Antisense a

Relative to the babA2 sequence under GenBank accession number AF033654

The detection of the babA2 gene was performed using polymerase chain reaction (PCR) targeting a 139-bp fragment of the central region of babA2 (Table 2). Four ng of DNA per ll of total reaction volume were used. The babA2 primers were designed on the basis of multiple alignments of 94 babA2 and 24 babB sequences that were retrieved from the GenBank sequence database. Primer selection was aimed at targeting conserved regions of the babA2 gene and at maximizing the number of nucleotide mismatches between primers and the non-targeted babB gene sequences. Primers were designed manually and evaluated and corrected using the Netprimer program (http:// www.premierbiosoft.com/netprimer/index.html); the specificity of the selected primers was verified using Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and MFEprimer-2.0 (http://biocompute.bmi.ac.cn/CZlab/ MFEprimer-2.0/index.cgi) web-based services. Three forward and one reverse primer selected for amplification of a 139-bp fragment of the babA2 gene are listed in Table 2. Misprinting sequence analyses showed that neither babB nor any other H. pylori sequences or human DNA are amplifiable with the selected primer combination. The babA2 PCR protocol was set up on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, USA) and performed using a HotStarTaqPlus DNA Polymerase kit (Qiagen, Hilden, Germany). The reaction mixture contained 100 ng (max 5 ll) of template DNA, 2.5 ll of 10 9 CoralLoad PCR Buffer with 15 mM MgCl2, 2 ll of extra 25 mM MgCl2, 0.5 ll of PCR Nucleotide MixPLUS (Roche Diagnostics, Manheim, Germany), 0.625 U of HotStarTaq Plus DNA polymerase, 0.4 lM of each primer, and water

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up to 25 ll. The cycling conditions used were 95 °C for 5 min, followed by 45 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min. This was followed by a final extension step at 72 °C for 10 min and cooling of the reaction mixture to 4 °C. The resulting amplicons from babA2 PCRs were analysed on a 2 % agarose gel and sequenced with the same primers as those used for PCR, as described previously (Platt et al. 2007). The obtained sequences were analysed and edited using the Vector NTI Advance v11.0 program (Invitrogen, Carlsbad, CA). The verification/confirmation of babA2 sequences was done with the ClustalW multiple alignment program of the BioEdit Sequence Alignment Editor v7.0.9.0 program package (North Carolina State University, Raleigh, NC), using the available H. pylori babA2 and babB sequences as standards for comparisons, and Blast analysis. In order to investigate whether newly developed primers have better sensitivity for detecting of babA2 in comparison to other BabA2 assays we compared the new primers to the most widely used primers for the babA2 gene detection, a subset of first 82 out of 163 biopsy samples was additionally tested, as described previously (Gerhard et al. 1999).

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Results Patient characteristics Out of 223 eligible children, 142 were girls and 81 were boys, with a mean age of 12.9 years (range, 3–18 years). The main indications for performing a gastroduodenoscopy were recurrent abdominal pain, diarrhea, chest pain, melena, weight loss and, rarely, vomiting. Two patients had ulcers, 19 gastric erosions and two bulbar erosions. Other patients had no visible signs of inflammation of the gastric mucosa. Detection of H. pylori and cagA, iceA, and vacA genotyping DNA was successfully extracted from biopsy specimens of all 58 patients enrolled during 2006–2010. The cagA, iceA, and vacA status was determined, as described previously (Homan et al. 2009). Out of 223 children included in the study (165 from the period 1999–2005 and 58 from the period 2006–2010) a total of 20 children (9.0 %) had multiple H. pylori genotypes and were excluded from further genotypephenotype correlation analyses. Histology

Statistical analysis The SPSS 21.0 statistical package (SPSS GmbH Software, Munich, Germany) was used for data management and analysis. Statistical significance of differences between groups was tested using the t test and Chi square test. A p value of less than 0.05 was considered statistically significant.

Ethical considerations None of the 223 clinical samples were collected solely for the purpose of this study. The study has been carried out in accordance with the code of Ethics of the World Medical Association (Declaration of Helsinki) and has been approved by the Ethics Committee of the Ministry of Health of Republic of Slovenia. Written informed consent was obtained from parents of all children included in the study.

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A total of 163 gastric mucosa tissue specimens from 203 patients infected with a single H. pylori genotype were available for histological analysis. In 57.1 % (93/ 163) of samples, the bacterial load was very high. The activity of inflammation was mostly mild (84/163, 51.5 %) and the degree of chronic inflammation was moderate (127/163, 77.9 %) in the majority of biopsies. Mild glandular atrophy was present in 51 of 163 samples (31.3 %), moderate in 2 of 163 biopsy samples (1.2 %) and there were no biopsies with severe glandular atrophy. Mild intestinal metaplasia was diagnosed in 9 specimens (5.5 %). Prevalence of the babA2 and cagA genes, vacA and iceA alleles Helicobacter pylori samples of all 163 children with valid histological assessment were tested for the presence of the babA2 gene. The babA2 gene was detected in 47.9 % (78/163) of samples. Direct PCRsequencing of the 139-bp amplicons confirmed the

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presence of babA2 sequences in all positive cases. In a subset of 82 samples, the babA2 gene was detected in 34.1 % (28/82) of samples when using primers designed by Gerhard et al. (1999) and in 50 % (41/ 82) of samples when using newly developed primers. Of the 163 H. pylori positive biopsy specimens, 92 (56.4 %) were cagA positive. The vacA s1 allele was found in 73.5 % (114/155) and vacA s2 allele in 26.5 % (41/155) of samples. The vacA m1 and m2 alleles were found in 41.3 % (64/155) and 58.7 % (91/ 155) of biopsy samples, respectively. Regarding the iceA gene, iceA1 predominated and was detected in 67.9 % (110/162) of isolates. The iceA2 allele was detected in 32.1 % (52/162) of samples. Association of the babA2 gene with gender and age groups The presence of the babA2 gene was not significantly associated with gender (p = 0.793; the statistical significance was tested using the Chi square test) and there were no age-related associations between the presence of the babA2 gene and any of the age groups (3–8, 9–13, 14–18 years, p = 0.276; Chi square test). Association of babA2 gene with cagA, vacA and iceA genotypes The relationships between the babA2 gene and other virulence genes were assessed for 152 out of 163 H. pylori isolates, which had complete genotyping results regarding cagA, vacA and iceA. The presence of the babA2 gene was strongly associated with the presence of cagA gene (p \ 0.001; Chi square test), vacA s1 allele (p \ 0.001; Chi square test) and vacA m1 allele (p \ 0.001; Chi square test). There were no statistically significant associations between the babA2 gene positivity and the presence of iceA1 or iceA2 and s2 or m2 alleles of vacA (Chi square test). Association of the babA2 gene with the presence of erosions/ulcer of the gastric mucosa The presence of the babA2 gene was correlated with children who had endoscopically detected lesions of gastric mucosa, because of H. pylori infection. After exclusion of children, who were infected with more than one H. pylori strain, erosive gastritis and ulcers were found in 19/152 children, with 7/19 lesions being

641 Table 3 Association between the presence of the babA2 gene determined by the 139-bp PCR assays and histological parameters No. of specimen

Total

Mean score

p value

0.001

Histological score 0

1

2

3

Bacterial density babA2 Positive Negative

0

2

22

54

78

2.67

1

10

35

39

85

2.32

Activity of inflammation babA2 Positive

2

34

34

8

78

1.62

Negative

3

50

29

3

85

1.38

0.023

Chronic inflammation babA2 Positive

0

4

60

14

78

2.13

Negative

0

11

67

7

85

1.95

0.017

Atrophy babA2 Positive

55

23

0

0

78

0.29

Negative

55

28

2

0

85

0.37

0.296

Intestinal metaplasia babA2 Positive Negative

76

2

0

0

78

0.03

78

7

0

0

85

0.08

0.107

babA2 negative. No association between the babA2 gene and the presence of erosive gastritis and ulcers was identified (p = 0,151; Chi square test). Association of the babA2 gene with histological parameters in gastric mucosa The correlation between the presence of babA2 and five histological parameters: (i) H. pylori density score; (ii) activity of inflammation; (iii) chronic inflammation of the gastric mucosa; (iv) presence of atrophy; and (v) the presence of intestinal metaplasia, is shown in Table 3. H. pylori strains, determined as babA2 positive by our PCR assay, were strongly associated with three out of five of the histological parameters studied: H. pylori density score (p \ 0.001; t test), activity of inflammation (p = 0.022; t test) and chronic inflammation of the gastric mucosa (p = 0.017; t test).

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Table 4 The babA, vacA s, vacA m and cagA genotype groups distribution

Discussion

Group

babA

vacA s

vacA m

Group 1

Positive

s1

Group 2

Positive

s1

Group 3

Positive

Group 4 Group 5

Adherence to epithelial cells of the gastric mucosa is crucial in the pathogenicity of H. pylori (Backert et al. 2011). The H. pylori genome contains more than 30 omp genes, of which the babA2 gene is the most important, encoding a protein that attaches bacteria to Lewisb epitopes, which are expressed on human epithelial cells, and enables the delivery of bacterial toxins near the gastric epithelium (Ilver et al. 1998). Since it has previously been shown that the primers widely used for babA2 gene detection might underestimate babA2 prevalence due to the location of the primers in a highly polymorphic zone of the babA2 gene (Gerhard et al. 1999; Torres et al. 2009; Fujimoto et al. 2007), a novel H. pylori babA2 gene-specific PCR assay was developed to assess the babA2 prevalence. The novel assay was developed on the basis of 118 publically available babA2 and babB sequences and employs a set of three forward and one reverse primer for specific amplification of a 139-bp fragment of the babA2 gene. The primers were selected in the central region of the babA2 gene, since it has previously been shown that babA and its paraloque babB are nearly identical in their 50 and 30 regions, with most of their sequence divergence being in their mid-regions (Pride et al. 2001). The prevalence of the babA2 gene among H. pylori infected adults varies from 32 to 72 % in western countries and from 39 to 100 % in Asia (Yamaoka 2008). A strong correlation between positive babA2 status and increased risk of developing significant gastro-duodenal disease was found in more than half of the published studies. These data also suggested geographic differences in the prevalence of the babA2 gene, with higher detection rates in Asian countries, and confirmed the important role of the babA2 gene in the pathogenesis of H. pylori related diseases. In contrast to adults, published data concerning the prevalence and clinical relevance of the babA2 gene in children are scarce and not consistent (Oleastro et al. 2003; Podzorski et al. 2003; Talarico et al. 2009; Garcia et al. 2010; Boyanova et al. 2010; Oleastro et al. 2010; Ozbey et al. 2013) (Table 1). As shown in Table 1, six studies were performed on a relatively small number of patients (ranging from 16 to 61), with babA2 positivity ranging from 17.2 to 84.4 %. In the present study, the babA2 gene was detected in 47.9 % of paediatric H. pylori isolates. Since the

cagA

No. of patients

m1

Positive

38

m1

Negative

7

s1

m2

Positive

19

Negative

s1

m1

Positive

13

Negative

s1

m2

Positive

11

Group 6

Negative

s1

m1

Negative

4

Group 7

Positive

s2

m2

Positive

1

Group 8

Positive

s1

m2

Negative

4

Group 9

Negative

s2

m2

Positive

2

Group 10

Negative

s1

m2

Negative

15

Group 11 Group 12

Positive Negative

s2 s2

m2 m2

Negative Negative

3 35

Association of combination of H. pylori genes with histological parameters A total of 152 strains with complete genotyping data for cagA, vacA s1, vacA m1, vacA s2, vacA m2 and babA status were identified, classified into 12 genotype groups (Table 4) and evaluated for the association with five different histological parameters of gastritis (Table 5). Group 1 strains (cagA, vacA s1, vacA m1 and babA2 positive), found in 38/152 (25 %) patients, group 12 strains (cagA, babA2 negative, vacA s2 and m2), found in 35/152 (23 %) patients, group 3 strains (cagA, vacA s1 and babA2 positive) found in 19/152 (12.5 %) patients, and group 4 strains (cagA, vacA s1 and m1 positive), detected in 13/152 (8.6 %) patients, represented the most common genotype combinations in Slovenian pediatric population. Group 1 strains were detected significantly more frequently among patients with high activity of inflammation and density score compared to other patients (p = 0.035 and p = 0.025, respectively; Chi square test). In addition, group 4 strains were also positively associated, but only with the activity of inflammation (p = 0.049; Chi square test). No correlation was found however between other H. pylori genotype groups and H. pylori density score, activity of inflammation, chronic inflammation of the gastric mucosa, the presence of atrophy or intestinal metaplasia.

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643

Table 5 Association between different groups of strains and five histological parameters of gastritis indicated as the p value (Chi square test) Group

Bacterial density

Activity of inflammation

Chronic inflammation

Atrophy

Intestinal metaplasia

Group 1

0.035

0.025

0.105

0.558

0.421

Group 2

0.315

0.897

0.847

0.990

0.939

Group 3

0.855

0.982

0.092

0.688

1

Group 4

0.049

0.801

0.388

0.646

0.983

Group 5

0.579

0.902

0.870

0.404

0.951

Group 6

0.790

0.895

0.672

0.988

0.001

Group 7

0.812

0.609

0.870

0.522

0.997

Group 8

0.014

0.895

0.566

0.582

0.973

Group 9

0.375

0.965

0.755

0.950

0.990

Group 10

0.037

0.470

0.001

0.942

0.819

Group 11 Group 12

0.149 0.310

0.970 0.004

0.265 0.290

0.100 0.050

0.184 0.999

prevalence of the babA2 gene in previous studies (Table 1) differed significantly, with the prevalences ranging from 17.2 to 84.4 % and with the use of different babA2 gene detection methods, it is difficult to compare the results and make definite conclusions. To further address this issue, a subset of 82 samples were additionally tested for the presence of the babA2 gene, using the most widely used primers, designed by Gerhard et al. (1999). While the babA2 gene was detected in 28/82 (34.1 %) of samples using the primers, designed by Gerhard et al. (1999), the newly developed primers detected additional 13 babA2 positive samples (41/82, 50 %), thus confirming that the novel PCR has generally a superior sensitivity for detection of the babA2 gene to the widely used PCR assay. In contrast, none of the samples, which tested babA2 negative using our novel PCR assay, was babA2 positive using Gerhard’s primers. However, the presence of the babA2 gene was not statistically associated with any of the age groups, suggesting that the older children are infected with equally pathogenic strains as the younger children. Our results are in agreement with a previous study by Oleastro et al. (2003), where no association between babA2 and age groups was found. The babA2 gene was also equally distributed among female and male patients and is therefore uniformly present in Slovenian pediatric population. To the best of our knowledge, the clinical relevance of babA2 in children has been studied to the date in

three studies only (Table 1). In our study population, a strong correlation was found between positive babA2 status and the density of H. pylori colonization, as well as the degree of active and chronic inflammation (Table 3). Our results are consistent with the great majority of published studies in adults (Olfat et al. 2005; Oleastro et al. 2003; Gerhard et al. 1999) and with the results of a recent study performed on 117 children from the Lisbon area (Oleastro et al. 2010), in which a significant correlation was established between the severity of gastric pathology and the presence of the babA2 gene. However, the results of our and Portuguese study disagree with studies performed on 47 US and 49 Turkish children (Talarico et al. 2009; Ozbey et al. 2013), suggesting possible geographic differences in the prevalence and clinical relevance of the babA2 gene. The lack of association between the presence of the babA2 gene and clinical findings in US and Turkish children could also be due to the small number of H. pylori isolates. In addition, is it possible that the reported geographical variation in babA2 prevalence is due, in full or in part, to geographical variation in the babA2 sequence causing differences in PCR detection rate. Therefore, a number of geographically diverse strains should be ideally included in the process of new primers design. Chronic infection with H. pylori can cause precancerous lesions in adults (Guarner et al. 2001). Atrophic gastritis and intestinal metaplasia are frequently observed in gastric biopsy specimens in adults but

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this relationship is still controversial in the pediatric population. The prevalence of gastric atrophy is not consistent among published pediatric studies, varying from 0 to 72 % (Dimitrov and Gottrand 2006). Gastric atrophy was observed in 32.5 % of our patients, mostly of mild degree. In this study, babA2 positive strains were not found more frequently in patients with gastric atrophy. Moreover, a positive association between the presence of the babA2 gene and intestinal metaplasia, which was detected in 5.5 % of patients, was also not confirmed in this study. These results suggest that other pathogenic factors might be responsible for the development of precancerous lesions in children, such as the presence of virulence genes other than babA2 or a longer duration of infection with H. pylori, known as the Correa cascade. The babA2 gene has previously been associated with several virulence factors, such as the cagA, vacA s1 and jhp0562 gene (Oleastro et al. 2003, 2010), suggesting that the co-expression of more virulent genes is a consequence of shared selective pressure, which enables H. pylori to adapt to the environment more efficiently. In the present study, the babA2 gene was similarly strongly associated with cagA, vacA s1 and m1 genotype, but not with other alleles of vacA gene and iceA1 or iceA2 gene. Furthermore, different distributions of H. pylori genotypes were observed in our cohort among children with high grade and low grade gastritis. Quadruple-positive strains (group 1) had the highest discriminating value for the detection of the degree of gastritis compared to the other strains. Therefore, the determination of cagA, vacA s1, vacA m1 and babA positive strains can improve the detection of children with severe gastric inflammation, which may be a result of a synergistic action of cagA, vacA s1, vacA m1 and babA gene products. The results of our study are in agreement with adult studies from Portugal, Germany, Finland and Cuba (Olfat et al. 2005; Torres et al. 2009), where differences in the prevalence of gene combinations were observed between ulcer and non-ulcer patients for the vacA s1, cagA and babA2 positive strains. To the best of our knowledge no data are available in literature concerning animal studies comparing babA2 positive mutant strains and level of gastric inflammation. Therefore, further studies are needed to clarify the exact pathogenicity role of babA2 gene also with the use of animal models and with in vitro studies.

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Antonie van Leeuwenhoek (2014) 106:637–645

There were some limitations in our study. First, the number of patients classified into the group 4 was too low. Ideally, group 1 strains (cagA, vacA s1, vacA m1 and babA2) should be compared with the larger number of group 4 strains (cagA, vacA s1, m1, babA2 negative) to prove the importance of virulence characteristics of the babA2 gene. Secondly, we might have underestimated the number of mixed infections due to the limited number of biopsy samples in the stomach and consequently some strains might have been undetected. Third, although the newly developed primers were designed to detect all babA2 gene positive samples on the basis of publically available babA2 sequences, the geographical differences might have led to the underestimation of the babA2 gene prevalence, thus further studies are needed. In conclusion, in the study which included the largest number of paediatric H. pylori samples to date, babA2 was detected in almost half of Slovenian children requiring gastroduodenoscopy. The babA2 status correlated positively with three out of five evaluated histological parameters: bacterial density score, activity of inflammation and chronic inflammation of the gastric mucosa. In addition, the combination of positive cagA, vacA s1, vacA m1 and babA alleles was significantly more frequent in group of children with high degree of gastritis compared to the rest of study population. This study confirmed that babA2 also plays an important role in the pathogenesis of H. pylori gastritis in the paediatric population, and not only in adults. Whether babA2 plays an important role in the development of atrophic gastritis and intestinal metaplasia in children remains unclear.

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Prevalence of the Helicobacter pylori babA2 gene and correlation with the degree of gastritis in infected Slovenian children.

The aims of our study were to determine the prevalence of the babA2 gene within Helicobacter pylori strains circulating in the Slovenian pediatric pop...
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