Virulence factors of Helicobacter pylori

To date a number of virulence factors have been identified and characterised from the gastric pathogen Helicobacter pylori. The vacuolating toxin (VacA) is a major determinant of H. pylori-associated gastric disease. In non-polarised cells, VacA alters the endocytic pathway, resulting in the release of acid hydrolases and the reduction of both extracellular ligand degradation and antigen processing. The toxin forms trans-membrane anion-specific channels and reduces the transepithelial electrical resistance of polarized monolayers. Localization of the VacA channels in acidic intracellular compartments causes osmotic swelling which, together with membrane fusion, leads to vacuole formation. The neutrophil-activating protein of H. pylori (HP-NAP) induces the production of oxygen radicals in human neutrophils via a cascade of intracellular activation events which may contribute to the damage of the stomach mucosa. This protein has recently been shown to be an important antigen in the human immune response to H. pylori infection. In addition, mice vaccinated with recombinant HP-NAP were protected against H. pylori challenge. H. pylori strains that are associated with severe tissue damage and inflammation possess the cag pathogenicity island that contains several genes encoding factors involved in the induction of proinflammatory cytokines/chemokines and of a type IV secretion system involved in the delivery of a highly immunogenic protein, CagA, into eukaryotic cells. Recent advances in our understanding of the involvement of VacA, HP-NAP and the CagA/Type IV secretion system in the H. pylori-associated disease process are discussed in this review.     

cagA, vacA and iceA Virulence Genes of Helicobacter pylori Isolates of Children in Finland

 cagA, vacA s and m genotypes and iceA alleles were analyzed from Helicobacter pylori strains isolated from 17 Finnish children and 32 children of non-Finnish origin living in Finland. Twelve children in the latter group were eastern European and 15 were of African origin. Only three children of non-Finnish origin were born in Finland. The vacA s1a subtype was more prevalent in the isolates from Finnish children than African children (76% vs. 7%, P<0.001);vacA s1b frequencies were 5% and 67%, respectively (P<0.001). The iceA1 allele was significantly more prevalent in African than Finnish isolates (93% vs. 35%, P<0.01). Considerable variation was noted in the frequency of vacA s1 subtypes and iceA alleles in children originating from different geographic regions, but the geographic variation of s1 subtypes resembled that described in other reports.     

Phenotypic Variation of Helicobacter pylori Isolates from Geographically Distinct Regions Detected by Lectin Typing

A total of 309 Helicobacter pylori isolates from 18 different countries were analyzed with a previously developed lectin typing system. The system was developed by using a proteolytic pretreatment to enhance the carbohydrate fraction of the sample. Four lectins from Ulex europaeus, Lotus tetragonolobus, Erythrina cristigali, and Triticum vulgaris were used to type the strains. The lectins were chosen for their specificities for sugars commonly encountered in the lipopolysaccharide of H. pylori. The isolates were received from their parent institutions as pellets of biomass and were typed at one of three centers (in Ireland, Sweden, and Estonia). All 16 possible lectin reaction patterns were observed in the study, with the isolates with the predominant pattern exhibiting reactions with all the lectins in the panel. For European patients suffering from gastritis, an association was noted between lectin reaction pattern MH4 and atrophic chronic gastritis; isolates with lectin reaction pattern MH4 were isolated from patients with atrophic chronic gastritis, whereas isolates with this pattern were not isolated from patients with chronic gastritis (P = 0.0006). In addition, statistically significant relationships were noted between the lectin reaction pattern and the associated pathology of isolates from the Swedish population. Isolates with patterns MH13 and MH16, which had low lectin reactivities, correlated with nonulcer disease (P = 0.0025 and P = 0.0002, respectively), and all four isolates from adenocarcinoma patients were characterized as possessing reaction pattern MH16. In contrast, isolates with lectin reaction patterns MH1 and MH10, which had high lectin reactivities, were associated with ulcer disease (P = 0.046 and P = 0.0022, respectively).     

Determination of Helicobacter pylori Virulence by Simple Gene Analysis of the cag Pathogenicity Island

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.     

Cytokine Expression in Pediatric Helicobacter pylori Infection

Helicobacter pylori infection is one of the most common gastrointestinal infections worldwide and almost invariably causes chronic gastritis in the infected host. A predominant Th1 profile has been demonstrated in H. pylori-infected mucosa from adults, but no previous study has evaluated in situ cytokine expression in children. We therefore examined expression of proinflammatory, anti-inflammatory, and regulatory cytokines by immunohistochemistry in cryopreserved antral biopsy specimens from 10 H. pylori-infected and 10 uninfected children and correlated expression of cytokines with histology scores. Concomitant expression of interleukin-8 (IL-8), gamma interferon (IFN-γ), IL-4, transforming growth factor β, and tumor necrosis factor alpha was seen in 8/10 H. pylori-infected cases and in 5/10 noninfected cases; all H. pylori-infected subjects showed staining for at least two of the cytokines. The proportion of epithelial cytokine-specific staining did not differ significantly between the groups, either in surface or glandular epithelium. Furthermore, no significant differences were noticed between intraepithelial or lamina propria lymphocyte staining in the groups. There was, however, a tendency of higher numbers of IFN-γ- and IL-8-positive cells in the H. pylori-infected group. IFN-γ and IL-8 lamina propria lymphocyte expression correlated significantly with antrum chronic inflammation, but there was no correlation between histology scores and epithelial cytokine expression. When the same techniques were used, the cytokine response appeared to be smaller in H. pylori-infected children than in adults, and there was no clear Th1 dominance. These results therefore suggest a different mucosal immunopathology in children. It remains to be determined whether the gastric immune response is downregulated in children with H. pylori infection and whether this is relevant to the outcome of infection.     

Molecular Basis of Pathogenicity in Helicobacter pylori Clinical Isolates

This study identified pathogenicity genes in 40 Helicobacter pylori clinical isolates. The cagA, vacA, and iceA genes were detected in 65%, 97.5%, and 97.5% of the isolates, respectively. The cagA, iceA1, and vacAs1a/m1 genes were related to erosive gastritis, whereas the vacAs2/m2 and iceA2 genes were associated with enanthematous gastritis.Helicobacter pylori is considered the major etiologic agent of chronic active gastritis, an essential catalyst in the emergence of peptic ulcer, and a risk factor for the development of gastric cancer (17). Studies indicate that the evolution of the infection depends in part on the expression of specific bacterial pathogenicity genes, such as cagA (cytotoxin-associated gene A), vacA (vacuolating cytotoxin), and iceA (induced by contact with epithelium) (2).The cagA gene is considered to be a marker for the presence of a cagA pathogenicity island (8). The cagA-positive H. pylori strains increase interleukin-8 production and gastric inflammation (5). The vacA gene encodes a vacuolating cytotoxin able to induce the formation of cytoplasmic vacuoles in epithelial cells (11). This gene comprises two variable regions: the signal region, with two alleles, s1 (subtypes s1a, s1b, and s1c) and s2, and the middle region, with the alleles m1 and m2 (3, 28). In general, the s1/m1 strains produce large amounts of vacuolating cytotoxin, the s1/m2 strains produce moderate amounts, and the s2/m2 strains produce little or none (3). The iceA gene has two alleles: iceA1 and iceA2. The iceA1 allele is associated with peptic ulcer, and iceA2 is related to asymptomatic gastritis (24, 29).This study analyzed the presence of cagA, vacA, and iceA genes in clinical isolates and correlated these findings with the endoscopic diagnosis. Forty isolates of H. pylori were obtained from biopsy specimens of the gastric antrum collected from dyspeptic patients admitted to the upper gastrointestinal endoscopic ward in the Hospital of the Federal University of Rio Grande, Rio Grande do Sul, Brazil. This study was approved by the ethics committee of our university. Informed consent was obtained from all patients.After collection, the biopsy specimens were kept in brain heart infusion broth (Acumedia, United States) with 20% glycerol and refrigerated (4 to 8°C) for a maximum of 4 h (22). This broth was thereafter vortexed, and 200 μl was added to medium Columbia agar (Oxoid, United Kingdom), supplemented with 7% sheep blood and with a selective mixture for Helicobacter species isolation (Cefar, Brazil). The agar plates were incubated under microaerophilic conditions (5 to 15% O₂ and 10% CO₂) at 37°C for 4 to 10 days (14). The identification of H. pylori was performed using catalase, oxidase, and urease tests, microscopy, and ureA gene detection (12, 19).The DNA extraction was performed after 48 h of bacterial growth. Colonies were collected and resuspended in 500 μl of 1× TE buffer. The suspension was centrifuged at 10,000 × g for 5 min, and the supernatant was thereafter discarded. The DNA from the clinical isolates was then extracted with DNAzol reagent (Invitrogen, United States) by the method of the manufacturer.The presence of the ureA, cagA, vacA, and iceA genes in the isolates was investigated by PCR using the primers described previously (6, 10, 21, 31). The PCR was performed as described by Rota et al. (for the ureA and cagA genes) and by Benenson et al. (for the alleles of the vacA and iceA genes) (4, 27).The statistical analysis was performed by using Fisher''s exact test, a chi-squared test, and a chi-squared test for linear trend. P values of less than 0.05 were considered statistically significant.The presence of the pathogenicity genes was studied in 40 clinical isolates of H. pylori. From those, 50% (20 of 40) were obtained from patients with endoscopic diagnosis of enanthematous gastritis and 50% (20 of 40) were obtained from patients with erosive gastritis.The cagA gene was identified in 65% (26 of 40) of the isolates. This frequency is similar to that found in previous studies of cagA in Brazil (14, 16, 18). The vacA and iceA genes were detected in 97.5% (39 of 40) of the samples. The vacAs1b (43.6%) and vacAm2 (53.9%) alleles were the most frequently detected in the 39 isolates, as well as the iceA2 allele (71.8%). This is an expected result, because these alleles have been reported in other studies (7, 18, 26). Moreover, 12.8% of the isolates verified the presence of the m1 and m2 alleles of the vacA gene, and 5.1% of the isolates had both iceA alleles. The detection of more than one allele of the middle region of vacA, as well as the identification of both iceA alleles in the same isolate, suggests coinfection of two different strains of H. pylori. Cases of patients being infected with multiple strains of H. pylori are not uncommon, being more frequent in areas of high H. pylori prevalence (9, 15, 23).The association between the cagA and vacA genes is described in Table ​Table1.1. All cagA-positive isolates confirmed the presence of vacA. The combinations vacAs1a/m1, vacAs1b/m1, vacAs1a/m2, vacAs1b/m2, and vacAs1b/m1m2 were present mainly in cagA-positive samples. A statistically significant association was observed between cagA and vacA (P < 0.001).

TABLE 1.

Association between the cagA gene and the allelic combinations of the vacA gene in isolates of H. pylori

vacA Genotypes and Genetic Diversity in Clinical Isolates of Helicobacter pylori

Genetic diversity in Helicobacter pylori strains may affect the function and antigenicity of virulence factors associated with bacterial infection and, ultimately, disease outcome. In this study, DNA diversity of H. pylori isolates was examined by analysis of vacA genotypes and by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (vacA, cagA, flaA, ureAB, and ureCD). Thirty-seven H. pylori isolates from 26 patients were successfully classified into distinct vacA allelic genotypes. The signal sequence allele s1 (31 of 37) predominated over the s2 allele (6 of 37) and was significantly associated with the occurrence (past or present) of gastric ulcers. A novel midregion allele, designated as m3, has been identified in two H. pylori isolates which could not be typed with midregion allele m1- or m2-specific primers. Additionally, significant nucleotide diversity yielding different amino acid sequences was demonstrated by DNA sequencing of vacA fragments from clinical isolates of H. pylori. Furthermore, RFLP analysis of 45 H. pylori isolates (including 15 paired isolates) obtained from antrum and corpus biopsy specimens from 30 individual patients showed remarkably high interhost diversity (one patient, one H. pylori strain) and intrahost identity in gene sequences coding for VacA, CagA, flagellin, and urease. Only in a single patient was a minor genotypic variation at different anatomic sites within the stomach identified. These data warrant the detailed analysis of the effect of genetic diversity on the function and antigenicity of H. pylori-associated virulence factors.

Helicobacter pylori is one of the most prevalent causes of infection in human beings worldwide. H. pylori persists in the human gastric mucus layer for decades and possibly for life, even in the face of a brisk humoral antibody response (3, 26). The extent of mucosal lesions induced by H. pylori varies, with only a small number of patients developing peptic ulceration and gastric cancer (3, 19). With the increasing emergence of antibiotic-resistant H. pylori strains (27), development of an effective vaccine may represent an alternative means of controlling or even preventing H. pylori infection (5, 21).Individual H. pylori isolates demonstrate a high level of genomic diversity as defined by different techniques, including conventional or pulsed-field gel electrophoresis and restriction endonuclease digestion of genomic DNA, PCR-amplified genomic DNA, individual genes, or ribosomal DNA (1, 9, 10, 14, 24). Genomic differences may affect virulence factors, altering their function and antigenicity. Antigenic variation of certain gene products may represent an immune escape mechanism for H. pylori strains in the host organism.Vacuolating cytotoxin (VacA) is a major H. pylori-associated virulence factor (25, 29), but it may also serve as a target for induction of a strong, long-lived, and effective anti-H. pylori-directed immune response (12, 18, 25). Although almost all H. pylori strains bear the vacuolating cytotoxin gene (vacA) and through expression of this gene produce an immunoreactive protein (2), only 50 to 60% exhibit detectable cytotoxin activity (6, 24). Hence, nonfunctional but immunogenic variants of VacA exist. It was recently reported that the 95-kDa protein isolated from culture supernatants of Tox⁻ strains (the Tox protein being a homolog of VacA) is recognized by an antiserum raised against the H. pylori cytotoxin from Tox⁺ strains but is not capable of inducing cell vacuolation (2). The humoral anti-VacA-directed immune response is predominantly directed against conformational epitopes of the cytotoxin (17). Therefore, the nucleotide or amino acid sequences of cytotoxin and its homolog provide the basis for the design of a genetically “detoxified” molecule which retains the structure and immunogenicity of the native protein.The level of in vitro cytotoxin activity appears to correlate with the clinical consequences of H. pylori infection. VacA functionality in turn correlates with specific vacA genotypes, defined by certain combinations of vacA signal sequences (s1a, s1b, and s2) and vacA midregion alleles (m1 and m2), including s1a-m1, s1b-m1, s1a-m2, s1b-m2, and s2-m2 (2). It is unknown whether these structural vacA differences impact on anticytotoxin-directed immunity.In this study, we employed a PCR-based method to define the vacA genotypes of 37 individual H. pylori isolates from antrum and corpus biopsy specimens obtained from 26 patients undergoing endoscopy. Genetic diversity was characterized in detail by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (i.e., vacA, cagA, flaA, ureAB, and ureCD) coding for virulence factors (VacA, CagA, flagellin, and urease). This analysis was complemented by sequence analysis of individual vacA and flaA genes.     

Outer Membrane Protein Expression Profile in Helicobacter pylori Clinical Isolates

The gram-negative gastric pathogen Helicobacter pylori is equipped with an extraordinarily large set of outer membrane proteins (OMPs), whose role in the infection process is not well understood. The Hop (Helicobacter outer membrane porins) and Hor (Hop-related proteins) groups constitute a large paralogous family consisting of 33 members. The OMPs AlpA, AlpB, BabA, SabA, and HopZ have been identified as adhesins or adherence-associated proteins. To better understand the relevance of these and other OMPs during infection, we analyzed the expression of eight different omp genes (alpA, alpB, babA, babB, babC, sabA, hopM, and oipA) in a set of 200 patient isolates, mostly from symptomatic children or young adults. Virtually all clinical isolates produced the AlpA and AlpB proteins, supporting their essential function. All other OMPs were produced at extremely variable rates, ranging from 35% to 73%, indicating a function in close adaptation to the individual host or gastric niche. In 11% of the isolates, BabA was produced, and SabA was produced in 5% of the isolates, but the strains failed to bind their cognate substrates. Interleukin-8 (IL-8) expression in gastric cells was strictly dependent on the presence of the cag pathogenicity island, whereas the presence of OipA clearly enhanced IL-8 production. The presence of the translocated effector protein CagA correlated well with BabA and OipA production. In conclusion, we found unexpectedly diverse omp expression profiles in individual H. pylori strains and hypothesize that this reflects the selective pressure for adhesion, which may differ across different hosts as well as within an individual over time.Helicobacter pylori persistently colonizes the stomachs of one-half of the world population and thereby causes gastric disorders and severe diseases, including active chronic gastritis and gastric or duodenal ulcer. Furthermore, H. pylori is considered a risk factor for the development of gastric cancer and mucosa-associated lymphoid tissue lymphoma (45). Ultrastructural studies of infected human gastric tissue revealed that a minor proportion of the bacteria are intimately attached to the epithelium of the gastric pits, whereas the majority of the H. pylori organisms actively move within the mucus overlying the gastric epithelium (22). Theoretical modeling of the dynamics of H. pylori infection identified adherence to the gastric mucosa as an important mechanism contributing to chronic colonization of the human stomach (7, 24).One characteristic feature of H. pylori is the high plasticity of its genome, which is caused by both an elevated mutation rate and an extensive exchange of genetic material, leading to free recombination of H. pylori genes (46, 50). The micro- and macrodiversity among H. pylori strains acts as an important driving force for adaptation to the hostile gastric environment and the variable living conditions during the inflammation process and/or during transmission between host individuals (32, 44). This genetic heterogeneity becomes especially apparent in the class of outer membrane protein (OMP) genes. The H. pylori genome contains more than 30 omp genes, which have been divided into the hop (helicobacter outer membrane porins) and hor (hop-related) groups (1). All H. pylori adhesins identified so far are in the Hop group of proteins (reviewed in references 3, 17, and 30).The blood group antigen binding adhesin (BabA) binds to ABO histo-blood group antigens and corresponding Lewis b antigens (Le^b), which are expressed in the human gastric mucosa of most individuals (23). A further H. pylori OMP, the sialic acid-binding adhesin (SabA), binds to sialylated carbohydrate structures, which are upregulated as part of complex gangliosides in inflamed gastric tissue. SabA was postulated to contribute to the chronic persistence of the infection (4, 29). Two highly homologous OMPs, the adherence-associated lipoproteins A and B (AlpA and AlpB), are also involved in H. pylori adherence to human gastric histo-tissue sections (35, 36), although a corresponding receptor for these proteins is not known. Interestingly, functional and especially intracellular signaling differences in AlpAB proteins between Western and East Asian H. pylori strains have been reported (28). BabB, BabC, and HP0227 are considered members of the bacterial adhesion family, since their genes belong to the hop group of H. pylori omp genes (1), but a function in adherence for these OMPs has not been demonstrated. In addition, H. pylori adherence to extracellular matrix proteins, including laminin and collagen type IV, has been described (48). H. pylori adherence to laminin was recently attributed to the binding of SabA to sialylated moieties on this molecule, whereas fibronectin binding was independent of the SabA and BabA proteins (49). The glycan structures on laminin have not yet been elucidated. It was thus unknown whether sialyl-dimeric Le^x is carried by this protein. However, SabA not only recognizes sialyl-dimeric Le^x but has also been shown to have a broader sialic acid recognition, including binding to α-2,3-linked Neu5NAc (49) that may be present on laminin (25). The intimate contact between H. pylori and the gastric epithelium enables the bacterium to manipulate signal transduction pathways, resulting in the induction of proinflammatory cytokines, such as interleukin-8 (IL-8), in epithelial cells (11). The IL-8 induction is attributable mainly to the action of the cag type IV secretion system, a 40-kb chromosomal locus, also called the cag pathogenicity island (cag-PAI), which is involved in translocation of the bacterial effector protein CagA into the host cells (34). In many H. pylori strains, the CagA protein itself is not directly involved in IL-8 induction (14), but for several strains a direct contribution of translocated CagA to NF-κB activation and IL-8 induction has been demonstrated (9). In addition, the outer inflammatory protein (OipA), which belongs to the large H. pylori OMP family, was shown to be involved in IL-8 induction in epithelial cells upon H. pylori infection (51, 52). In another study, we reported that an oipA mutant strain induced similar IL-8 values to those in the parental wild-type (wt) strain (33, 51). Thus, the role of the OipA protein in this process remains to be elucidated in more detail.Another H. pylori OMP-encoding gene, hopQ, exists as two highly divergent alleles. The type I hopQ allele is mainly found associated with East Asian H. pylori strains, in close association with the cagA gene, whereas the type II hopQ allele is commonly found in Western H. pylori strains lacking cagA (10). Interestingly, certain HopQ OMPs are able to attenuate H. pylori adherence to gastric epithelial cells and thereby affect the efficiency of CagA translocation into epithelial cells (27). Thus, the expression pattern of H. pylori omp genes is expected to have an important influence on H. pylori adaptation and virulence.In order to obtain the first more global overview of the complexity of H. pylori omp gene expression, we concentrated on a set of eight different OMPs and CagA. No data about their expression or possible adhesive or other functions were available, but all selected omp genes belong to the adhesin-encoding hop gene family. Furthermore, we chose our set of 200 clinical H. pylori isolates from a pediatric clientele and tried to correlate the OMP profile and the cag status with adherence properties of the strains and their capacity to induce IL-8 in epithelial cells.     

cagA-Positive Helicobacter pylori Populations in China and The Netherlands Are Distinct

The aim of this research was to study whether and to what extent Chinese cagA-positive Helicobacter pylori isolates differ from those in The Netherlands. Analysis of random amplified polymorphic DNA (RAPD)-PCR-assessed DNA fingerprints of chromosomal DNA of 24 cagA-positive H. pylori isolates from Dutch (n = 12) and Chinese (n = 10) patients yielded the absence of clustering. Based on comparison of the sequence of a 243-nucleotide part of cagA, the Dutch (group I) and Chinese (group II) H. pylori isolates formed two separate branches with high confidence limits in the phylogenetic tree. These two clusters were not observed when the sequence of a 240-bp part of glmM was used in the comparison. The number of nonsynonymous substitutions was much higher in cagA than in glmM, indicating positive selection. The average levels of divergence of cagA at the nucleotide and protein levels between group I and II isolates were found to be high, 13.3 and 17.9%, respectively. Possibly, the pathogenicity island (PAI) that has been integrated into the chromosome of the ancestor of H. pylori now circulating in China contained a different cagA than the PAI that has been integrated into the chromosome of the ancestor of H. pylori now circulating in The Netherlands. We conclude that in China and The Netherlands, two distinct cagA-positive H. pylori populations are circulating.     

Distinct B-cell clonal bands in Helicobacter pylori gastritis with lymphoid hyperplasia

Helicobacter pylori (Hp)-associated gastritis is a risk factor for gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Clonal B-cell populations are present in both reactive and neoplastic MALT tissue, thus limiting their usefulness in the evaluation of gastric lymphoid infiltrates in endoscopic biopsy specimens. The aim of this study was to identify the presence of clonal B-cell populations in Hp-gastritis with MALT and to assess their usefulness in distinguishing reactive from malignant infiltrates. Routinely fixed paraffin-embedded blocks from 20 patients with Hp-gastritis with lymphoid hyperplasia were analysed for B-cell clonality by a semi-nested polymerase chain reaction (PCR) using FRIII/LJH and FRIII/VLJH primers for amplification of the VDJ region of the immunoglobulin heavy chain gene. The histopathological findings were evaluated according to a previously published scoring system. Immunohistochemistry was performed by the labelled streptavidin-biotin technique using the following primary antibodies: CD45, CD45RO, CD3, CD20, and cytokeratin. The histopathological findings were diagnostic of Hp-chronic active gastritis (grade 2, n=17; grade 3, n=3). Scattered intraepithelial B-cells were present in all cases and non-destructive lymphoepithelial lesions in one grade 3 case. Amplifiable DNA was obtained from all samples. Clonal bands were observed in ten (7/17 grade 2 and 3/3 grade 3 lesions) and polyclonal smears in ten cases (all grade 2). The clonal bands were often (n=6) associated with a background polyclonal smear and were not reproducible from deeper sections (n=10) or another paraffin block (n=1), while the clonal bands in control low-grade MALT lymphomas were not associated with a background smear and were reproducible from deeper sections. None of the patients has developed lymphoma to date (follow-up 21-44 months). In conclusion, B-cell clonal bands are common in H. pylori-gastritis with lymphoid hyperplasia. The irreproducibility of these bands is a useful feature in favouring a reactive process.     

Virulence markers of Helicobacter pylori in patients with diarrhoea-dominant irritable bowel syndrome

Recent studies suggest that irritable bowel syndrome (IBS) is associated with low-grade inflammation. This study aims to determine the distribution of Helicobacter pylori cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) alleles (e.g., s1 and s2) in patients with diarrhoea-dominant IBS (IBS-D) as the latter causes vacuolation in colonic epithelial cells in vitro. One hundred and seventy patients meeting Rome III criteria for IBS-D (mean age: 40 +/- 15 years) were enrolled. Gastric biopsy was assessed histologically and DNA extraction was performed by polymerase chain reaction (PCR) for H. pylori genus 16S ribosomal DNA (16S rDNA), cagA and vacA allele s1 and s2. There was no age- or gender-related difference in H. pylori positivity in IBS-D compared to the control group. H. pylori was positive in 116 (68%) with IBS-D compared to 88 (55%) in the control group (P=0.01). cagA was positive in 73 (63%) with IBS-D compared to 42 (48%) in the control group (P=0.03). vacA s1 was positive in 61 (53%) with IBS-D compared to 32 (36%) in the control group (P=0.02). cagA s1 was positive in 39 (34%) with IBS-D compared to 13 (15%) in the control group (P=0.002).     

Virulence factor genotypes of Helicobacter pylori affect cure rates of eradication therapy

The cure rates of Helicobacter pylori infection by using a combination of a proton pump inhibitor (PPI) and antimicrobial agents are mainly influenced by bacterial susceptibility to antimicrobial agents and the magnitude of acid inhibition during the treatment. Currently used empirical triple therapies do not reliably produce a ≥80% cure rate on an intention-to-treat basis. Therefore, tailored regimens based on relevant microbiological findings and pharmacogenomics are recommended for attaining an acceptable ≥95% cure rate. Recently, virulence factors of H. pylori, such as cagA and vacA, are reported to be major factors determining the cure rates. Individuals infected with strains with cagA-negative and vacA s2 genotypes have significantly increased risk of eradication failure of H. pylori infection. These virulence factors enhance gastric mucosal inflammation and are associated with the development of peptic ulcer and gastric cancer. H. pylori virulence factors induce proinflammatory cytokines, such as interleukin (IL)-1, IL-8, and tumor necrosis factor (TNF)-  which influence mucosal inflammation and/or gastric acid secretion. When physicians select an H. pylori eradication regimen with an acceptable cure rate, they might need to consider H. pylori virulence factors, especially cagA and vacA.     

Secondary Resistance Among 554 Isolates of Helicobacter pylori After Failure of Therapy

 In a study to determine secondary resistance among Helicobacter pylori isolates, gastroenterologists from several German cities submitted over a 3-year period to centre A (Regensburg) or centre B (Freiburg) gastric biopsies from patients in whom one or more therapies to eradicate Helicobacter pylori had failed. Rates of resistance among the collections of 302 (centre A) and 252 (centre B) isolates were, respectively, as follows: to metronidazole, 75% and 66%; to clarithromycin, 58% and 49%; to amoxicillin, 0%; to ciprofloxacin, 9%; to doxycycline, 0%; and to rifampin, 0%. Resistance to clarithromycin was associated with metronidazole resistance in 89% and 85% of the isolates in centre A and centre B, respectively.     

Molecular Characterisation of Virulent Gene vacA in Helicobacter pylori Clinical Isolates from Patients with Gastroduodenal Diseases in Assam,India

Background: Helicobacter pylori, the gastric bacterium, is widely known to be one of the most genetically diverse group of organisms whose pathogenesis as well as the diversity in infection outcome may be attributed to a variety of virulent genes. Aim: This study aimed to study the molecular profile of H. pylori vacA gene by determining the phylogenetic relatedness and genetic diversity of the strains isolated in this region with those of other geographical regions. Materials and Methods: A total of twenty H. pylori clinical strains were isolated from randomly selected 100 patients suffering from gastroduodenal diseases as well as endoscopically normal patients in a cross-sectional hospital-based setting from January 2016 to May 2017. VacA signal sequence and mid regions of H. pylori were amplified by polymerase chain reaction followed by DNA sequencing and phylogenetic analysis. Results: VacA s1m1 allelic variant was more prevalent in our study, regardless of the clinical outcomes. Phylogenetic analysis of VacA s1 strains revealed clustering of most of the strains. VacA m1 strains clustered with Bangladesh strains which is a country nearest to India. Conclusion: Prevalence of VacA s1m1 strains may account for high risk of transmission of this gastric pathogen and the overall risk of acquiring infection. Phylogenetic analysis results suggests the prevalence of high genetic diversity in our region. Our findings may aid in developing a better understanding of the genetic structure of H. pylori and the pathophysiology of associated diseases, thus facilitating the implementation of various treatment options.     

Virulence factors of Spanish Helicobacter pylori isolates and correlation with gastritis or ulcer production

Objective: To study the cagA , vacA and iceA status of Helicobacter pylori clinical isolates obtained from adult patients suffering from peptic ulcer or gastritis in order to find if these virulence factors are useful in determining a strain to be a gastritis or ulcer producer.
Methods: One hundred and five H. pylori strains from patients with gastritis and ulcer were studied. Culture and identification was done by standard methodology. cagA, vacA and iceA detection was performed by PCRs previously described. Results were visualized by agarose gel electrophoresis.
Results: Amplified fragments of 297 bp ( cagA ), 259 bp ( vacA s1), 286 bp ( vacA s2) and 975 bp ( iceA ) were detected. cagA was detected in 83.3% and 91.3% of gastritis and ulcer strains respectively (p>0.05). s1 was detected in 57.1% of gastritis strains and 62.3% of ulcer strains (p>0.05). cagA was strongly related with the s1 allele. iceA was more prevalent in strains from gastritis (82.4% versus 66.7%). The combination of cagA, vacA and iceA was not correlated with the production of peptic ulcer disease.
Conclusions: These data suggest that the combination of cagA, vacA and iceA cannot be used to predict severe gastric disease in Spanish H. pylori clinical isolates.     

Distinct variants of Helicobacter pylori cagA are associated with vacA subtypes.

The diversity of the cytotoxin-associated gene (cagA) of Helicobacter pylori was analyzed in 45 isolates obtained from nine countries. We examined variation in the 5' end of the cagA open reading frame as determined by PCR and sequencing. Phylogenetic analysis revealed the existence of at least two distinct types of cagA. One variant (cagA1) was found exclusively in strains from Europe, the United States, and Australia, whereas a novel variant (cagA2) was found in strains from East Asia. The greatest diversity between cagA1 and cagA2 was found in the first 20 amino acids of the cagA open reading frame, where several consistent insertions or deletions were observed. Additional cagA sequence variants that could be classified as separate subtypes were found in two of three Peruvian and in five of seven U.S. strains tested. The calculated isoelectric point of the first 154 amino acids of the cagA1 variants (7.52 +/- 1.54) was significantly higher than that of the first 154 amino acids of the cagA2 variants (5.61 +/- 0.94; P < 0.001). Most cagA2 strains contained vacA subtype s1c (P < 0.001), and in vacA m1 strains cagA1 was more frequently observed than cagA2. These results show the epidemiological relationship between cagA and vacA at the subtype level and indicate the existence of distinct H. pylori lineages that are not uniformly distributed over the globe.     

Host Cell Responses to Genotypically Similar Helicobacter pylori Isolates from United States and Japan

Associations of Helicobacter pylori genotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-three cagA⁺ vacAs1/m1 H. pylori isolates (United States, n = 24; Japan, n = 39) and eight cagA-negative vacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/or cag island mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and Japanese cagA⁺ vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frame oipA open reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus, H. pylori cagA⁺ vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by the cag island in vitro. Assessment of apoptosis in vitro identified a group of H. pylori isolates that induce more severe gastric inflammation and atrophy.