Comparing genomes of Helicobacter pylori strains from the high-altitude desert of Ladakh, India

The genomic diversity of Helicobacter pylori from the vast Indian subcontinent is largely unknown. We compared the genomes of 10 H. pylori strains from Ladakh, North India. Molecular analysis was carried out to identify rearrangements within and outside the cag pathogenicity island (cag PAI) and DNA sequence divergence in candidate genes. Analyses of virulence genes (such as the cag PAI as a whole, cagA, vacA, iceA, oipA, babB, and the plasticity cluster) revealed that H. pylori strains from Ladakh are genetically distinct and possibly less virulent than the isolates from East Asian countries, such as China and Japan. Phylogenetic analyses based on the cagA-glr motifs, enterobacterial repetitive intergenic consensus patterns, repetitive extragenic palindromic signatures, the glmM gene mutations, and several genomic markers representing fluorescent amplified fragment length polymorphisms revealed that Ladakhi strains share features of the Indo-European, as well as the East Asian, gene pools. However, the contribution of genetic features from the Indo-European gene pool was more prominent.     

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.     

Experimental Infection of Mongolian Gerbils with Wild-Type and Mutant Helicobacter pylori Strains

Experimental Helicobacter pylori infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry cagA (cagA-positive or cagA-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic ureA, cagA, or vacA mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after ≥20 in vitro passages of wild-type strain G1.1 or with the ureA mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the cagA (25 of 25) or vacA (25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human cagA-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human cagA-negative isolates (P < 0.001). Similar to infection in humans, H. pylori colonization of gerbils induced gastric inflammation and a systemic antibody response to H. pylori antigens. These data confirm the utility of gerbils as an animal model of H. pylori infection and indicate the importance of bacterial strain characteristics for successful infection.     

Detection of Helicobacter pylori cagA gene in gastric biopsies, clinical isolates and faeces

Purpose: Helicobacter pylori infection is common in the developing countries. The cagA gene is a marker of pathogenicity island (PAI) in H. pylori. The aim of this study was to determine the prevalence of cagA among dyspeptic patients in Bahrain directly from gastric biopsy and stool specimen. Methods: A total of 100 gastric biopsy samples, 16 clinical isolates and 44 faecal specimens were collected from Bahraini adult dyspeptic patients. cagA gene of H. pylori was assessed using polymerase chain reaction (PCR). Results: The cagA gene was detected in 59 (59%) from biopsy specimens, 10 (62%) clinical isolates and in 10 (22.7%) faecal specimens. The detection of cagA positive H. pylori was significantly higher in patients with duodenal ulcer (80%) compared to those with other endoscopic finding (42%) (P<0.05). Conclusions: Using PCR to detect cagA gene directly from biopsy is a rapid and reliable technique. However, using stool specimen for genotyping in our patients showed reduced sensitivity.     

Typing of Helicobacter pylori vacA Gene and Detection of cagA Gene by PCR and Reverse Hybridization

The present report describes an analysis of two virulence genes of Helicobacter pylori. Parts of the cagA gene, as well as parts from the signal (s) and middle (m) regions of the mosaic vacA gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the vacA s region (s1a, s1b, and s2 alleles), the vacA m region (m1 and m2 alleles), and the cagA gene. A total of 103 H. pylori-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (n = 55) and The Netherlands (n = 48) were tested by the PCR-LiPA. cagA was detected in 84 and 73% of the Portuguese and Dutch patients, respectively. vacA typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type s1b, whereas most Dutch patients (61%) contained type s1a (P < 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (P = 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of vacA type s1 (s1a or s1b; P = 0.008) and cagA (P = 0.003) genes.     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

New approaches for genotyping of Helicobacter pylori based on amplification of polymorphisms in intergenic DNA regions and at the insertion site of the cag pathogenicity island

The population of the gastric pathogen Helicobacter pylori shows a high degree of genetic diversity. It is well established that heterogeneity at the isolate level is caused by nucleotide transitions within genes, differences in the gene order, and by genetic instability of single genes as well as of a large virulence-associated genomic DNA region, the cag pathogenicity island (PAI). Analysis of intergenic regions with specific PCR-assays developed in this study, revealed that DNA polymorphisms in the noncoding DNA localized in front of the genes ribA and vacA and at the insertion site of the cag PAI contribute to the genetic diversity of H. pylori and are useful for differentiation of individual isolates. Thirteen individual genotypes were identified by PCR analysis of these polymorphic loci in 487, 241, and 182 clinical H. pylori isolates. Sequence analysis revealed that genetic variability in front of genes ribA and vacA, and in the intergenic region at the PAI insertion site is caused by insertion and deletions of so-far-unknown DNA sequences as well as by parts of the H. pylori IS elements IS605 and IS606, respectively. The new genotypes identified could be used to differentiate antrum and corpus isolates from the same patients. Their combination with vacA allele subtypes and with the cagA status allowed to differentiate 140 isolates in 51 subtypes. In 36 cases the corresponding genotype patterns were isolate specific. In summary, the results confirm that DNA polymorphisms in intergenic regions contribute to the genetic diversity of H. pylori. Although individual H. pylori genotypes were not associated with peptic ulcer disease, the PCR-based approaches for their detection developed here should be of use for further investigation of genetic diversity in H. pylori and for epidemiological purposes. Received: 20 June 2000     

Variants of the 3′ Region of the cagA Gene in Helicobacter pylori Isolates from Patients with Different H. pylori-Associated Diseases

The CagA protein of Helicobacter pylori is an immunogenic antigen of variable size and unknown function that has been associated with increased virulence as well as two mutually exclusive diseases, duodenal ulcer and gastric carcinoma. The 3′ region of the cagA gene contains repeated sequences. To determine whether there are structural changes in the 3′ region of cagA that predict outcome of H. pylori infection, we examined 155 cagA gene-positive H. pylori isolates from Japanese patients including 50 patients with simple gastritis, 40 with gastric ulcer, 35 with duodenal ulcer, and 30 with gastric cancer. The 3′ region of the cagA gene was amplified by PCR followed by sequencing. CagA proteins were detected by immunoblotting using a polyclonal antibody against recombinant CagA. One hundred forty-five strains yielded PCR products of 642 to 651 bp; 10 strains had products of 756 to 813 bp. The sequence of the 3′ region of the cagA gene in Japan differs markedly from the primary sequence of cagA genes from Western isolates. Sequence analysis of the PCR products showed four types of primary gene structure (designated types A, B, C, and D) depending on the type and number of repeats. Six of the seven type C strains were found in patients with gastric cancer (P < 0.01 in comparison to noncancer patients). Comparison of type A and type C strains from patients with gastric cancer showed that type C was associated with higher levels of CagA antibody and more severe degrees of atrophy. Differences in cagA genotype may be useful for molecular epidemiology and may provide a marker for differences in virulence among cagA-positive H. pylori strains.     

The cag PAI is intact and functional but HP0521 varies significantly in Helicobacter pylori isolates from Malaysia and Singapore

Helicobacter pylori-related disease is at least partially attributable to the genotype of the infecting strain, particularly the presence of specific virulence factors. We investigated the prevalence of a novel combination of H. pylori virulence factors, including the cag pathogenicity island (PAI), and their association with severe disease in isolates from the three major ethnicities in Malaysia and Singapore, and evaluated whether the cag PAI was intact and functional in vitro. Polymerase chain reaction (PCR) was used to detect dupA, cagA, cagE, cagT, cagL and babA, and to type vacA, the EPIYA motifs, HP0521 alleles and oipA ON status in 159 H. pylori clinical isolates. Twenty-two strains were investigated for IL-8 induction and CagA translocation in vitro. The prevalence of cagA, cagE, cagL, cagT, babA, oipA ON and vacA s1 and i1 was >85%, irrespective of the disease state or ethnicity. The prevalence of dupA and the predominant HP0521 allele and EPIYA motif varied significantly with ethnicity (p < 0.05). A high prevalence of an intact cag PAI was found in all ethnic groups; however, no association was observed between any virulence factor and disease state. The novel association between the HP0521 alleles, EPIYA motifs and host ethnicity indicates that further studies to determine the function of this gene are important.     

Lack of correlation between vacuolating cytotoxin activity,cagA gene inHelicobacter pylori, and peptic ulcer disease in children

To determine the prevalence of thecagA gene and vacuolating cytotoxin inHelicobacter pylori isolates obtained from children and to characterize the relationship betweencagA, cytotoxin production, and ulcerogenesis, pediatricHelicobacter pylori isolates were tested forcagA by the polymerase chain reaction and for vacuolating cytotoxin by a cell culture assay.Helicobacter pylori isolates were obtained from 33 children referred for upper gastrointestinal endoscopy. Twenty-six of these isolates were tested forcagA by the polymerase chain reaction; all 26 (100%) were positive. Of the 26 children from whom these isolates were obtained, 26 (100%) had chronic gastritis and 12 (46%) had duodenal ulcers. Nine (30%) of 30 isolates tested showed expression of vacuolating cytotoxin, only three of which came from patients with duodenal ulceration (odds ratio 0.81, 95% confidence interval 0.1–5.3). Of the 23cagA-positive isolates tested for cytotoxin, only nine (39%) were positive. There was no association between vacuolating cytotoxin and clinical symptoms, nor was cytotoxicity associated with ulcerogenesis. In summary, the findings suggest thatcagA is not a marker of duodenal ulceration or of vacuolating cytotoxin production in children referred for endoscopy.     

Diphenyleneiodonium Inhibits Apoptotic Cell Death of Gastric Epithelial Cells Infected with Helicobacter pylori in a Korean Isolate

NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacter pylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediate apoptotic cell death, direct involvement of NADPH oxidase on H. pylori-induced apoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reported as cagA⁺, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelial AGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability, hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the development of gastric inflammation associated with H. pylori infection by suppressing abnormal apoptotic cell death of gastric epithelial cells.     

Analysis of Clarithromycin Resistance and CagA Status in Helicobacter pylori by Use of Feces from Children in Thailand

The clarithromycin resistance and CagA status of Helicobacter pylori in Thai children were investigated using fecal samples. Of the 284 samples, H. pylori was detected in 120 samples, and the clarithromycin resistance rate was 29.2%. The cagA gene was detected in 59 samples, and only 6.8% of these samples contained the East Asian CagA type.Helicobacter pylori is a pathogenic bacterium that colonizes the human stomach. The prevalence of antibiotic-resistant H. pylori, especially clarithromycin-resistant H. pylori, has been increasing worldwide and makes it difficult to successfully eradicate H. pylori. Clarithromycin resistance in H. pylori has been shown to be due to mutations at positions 2142 and 2143 of the 23S rRNA gene (7, 9).Although H. pylori is closely associated with gastric cancer, the rate of mortality due to gastric cancer is relatively low in Thailand, even though the rate of H. pylori infection in Thailand has been reported to be over 80% (6, 8). A difference in pathogenicity between H. pylori strains may explain the lack of the expected correlation between the rate of mortality due to gastric cancer and the rate of H. pylori infection. The CagA protein, which is one of the most important pathogenicity factors of H. pylori, has been classified into two types: the East Asian CagA type, found in H. pylori isolates from Japan, South Korea, and China, and the Western CagA type, found in H. pylori isolates from Europe, North America, and Australia. Each CagA type has tyrosine phosphorylation segments characterized by a Glu-Pro-Ile-Tyr-Ala (EPIYA) motif in the C-terminal region (3). However, the Western CagA type contains the EPIYA-A and EPIYA-B segments, followed by a variable number of EPIYA-C segments, while the East Asian CagA type contains the EPIYA-A, EPIYA-B, and EPIYA-D segments. Furthermore, the East Asian CagA type has been reported to induce more-severe cellular changes than the Western CagA type (2).Recently, we identified a noninvasive method for detecting clarithromycin-resistant H. pylori isolates from feces with high sensitivity and specificity (5). In this study, we used this previously developed method to investigate the clarithromycin resistance and CagA status of H. pylori in feces of Thai children.Fecal samples were obtained from 284 children (116 males/116 females; mean age, 6.60 years [range, 1 to 12 years]) from three schools in Chiang Mai in August 2006. The ages and genders of 52 children of ethnic minorities could not be obtained. The study protocol and the informed-consent document were reviewed and approved by Research Ethics Committee, Faculty of Medicine, Chiang Mai University.DNA was extracted from the feces, and the 23S rRNA gene of H. pylori was amplified as previously described (5). Samples were considered to contain clarithromycin-resistant H. pylori if mutations at positions 2142 and 2143 of the 23S rRNA gene were detected.For the amplification of the cagA gene, we designed new primers targeting the region containing the EPIYA-A and EPIYA-B segments by comparing 81 of the cagA genes registered in the DNA Data Bank of Japan (data not shown). Amplification was performed using primer pairs comprising primers 2553F (5′-AACCCTAGTCGGTAATGGGTTRTCT-3′) and 3222R (5′-ATTGCTATTAATGCGTGTGTGGC-3′) for the first-round PCR and 2612F (5′-CGGACATCAGGAAAGAATTGAA-3′), 2609F (5′-TTTCGGATATCAAGAAGAATTGAA-3′), and 2998R (5′-TTGAAAGCCCTACTTTACTGAGATCA-3′) for the second-round PCR.Samples were designated cagA positive when a PCR product of 180 bp was detected after the third-round PCR, which was performed using Go Taq Green master mix (Promega, Madison, WI) and the 2609F, 2612F, 2779R (5′-CACTCACCTTTTTTAGCAACTTGAG-3′), and 2780R (5′-GCTTTTACCTTTTTAGCAACTTGAG-3′) primers. The cagA-typing PCR was performed using an East Asian CagA-specific primer pair (East-Asian-F [5′-AAAGGAGTGGGCGGTTTCA-3′] and East-Asian-R [5′-CCTGCTTGATTTGCCTCATCA-3′]) and a Western CagA-specific primer pair (Western-F [5′-GGCATGATAAAGTTGATGATCTCAGT-3′] and Western-R [5′-AAAGGTCCGCCGAGATCAT-3′]), which targeted the EPIYA-D and EPIYA-C segments, respectively. Each typing PCR was performed using 1.5 μl of the second PCR product. For each PCR amplification, a PCR mixture that contained ultrapure water as the template was included to rule out false-positive results.Of the 284 fecal samples obtained from Thai children, H. pylori was detected in 120 (42.3%). Of the 120 H. pylori-positive samples, clarithromycin-resistant H. pylori was detected in 35 (29.2%) samples, and both clarithromycin-susceptible and -resistant H. pylori isolates were detected simultaneously in 5 samples. The incidence of clarithromycin-resistant H. pylori in this study was slightly higher than the rate reported for Thai adults in other studies (23.2%) (4). Because there have been few studies that focused on the rate of clarithromycin-resistant H. pylori in Thai children, the results of this study will be useful for estimating the rate of clarithromycin-resistant H. pylori infection in adults in Thailand in the future.The cagA gene was present in 59 (49.2%) of the H. pylori-positive samples. Of these samples, 20 (33.9%) samples contained the Western CagA type (containing the EPIYA-C segments) and 4 (6.8%) contained the East Asian CagA type (containing the EPIYA-D segments). The remaining 35 samples, which lacked any EPIYA-C or EPIYA-D motifs, were considered to contain the Western CagA type (1). To confirm the absence of the EPIYA-C and EPIYA-D segments in these 35 samples, DNA sequencing was performed on 14 cagA genes by using the second PCR product, and none of the cagA genes analyzed had an EPIYA-C or EPIYA-D segment. In summary, H. pylori isolates containing the East Asian CagA type (6.8%) were significantly less prevalent than H. pylori isolates containing the Western CagA type (93.2%). Most of the H. pylori strains isolated in Asian countries with high incidences of deaths from gastric cancer, such as Japan and China, have been reported to contain the East Asian CagA type (10). Since differences in the prevalences of the East Asian CagA type in Asian countries have been suggested to be one of the reasons underlying the differential mortality rates associated with gastric cancer (2), further investigation is needed to confirm this possibility.In this study, we detected a high proportion (59.3%) of CagA that contained neither the EPIYA-C nor the EPIYA-D segment. The low incidence of Western CagA containing the EPIYA-C segment in Thailand may be one of the reasons for the low mortality rate associated with gastric cancer in Thailand.In conclusion, we show that the prevalence of the CagA types of H. pylori in Thai children differs from that reported for other Asian countries. Furthermore, our study demonstrates the usefulness of this approach for detecting and typing CagA in H. pylori by using feces. This method may prove useful in further investigations of the prevalences of the CagA types in H. pylori isolates from infected individuals.     

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.     

Clinical relevance of cagA and vacA gene polymorphisms in Helicobacter pylori isolates from Senegalese patients

The molecular epidemiology of Helicobacter pylori in Africa is poorly documented. From January 2007 to December 2008, we investigated 187 patients with gastric symptoms in one of the main tertiary hospitals in Dakar, Senegal. One hundred and seventeen patients were culture-positive for H. pylori. Polymorphisms in vacA and cagA status were investigated by PCR; the 3′-region of cagA was sequenced, and EPIYA motifs were identified. Bacterial heterogeneity within individuals was extensively assessed by using an approach based on vacA and cagA heterogeneity. Fourteen per cent of H. pylori-positive patients displayed evidence of mixed infection, which may affect disease outcome. Patients with multiple vacA alleles were excluded from subsequent analyses. Among the final study population of 105 patients, 29 had gastritis only, 61 had ulcerated lesions, and 15 had suspicion of neoplasia based on endoscopic findings. All cases of suspected neoplasia were histologically confirmed as gastric cancer (GC). The cagA gene was present in 73.3% of isolates. CagA proteins contained zero (3.7%), one (93.9%) or two (2.4%) EPIYA-C segments, and all were western CagA. Most of the isolates possessed presumed high-vacuolization isotypes (s1i1m1 (57.1%) or s1i1m2 (21.9%)). Despite the small number of cases, GC was associated with cagA (p 0.03), two EPIYA-C segments in the C-terminal region of CagA (p 0.03), and the s1 vacA allele (p 0.002). Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of GC in Senegal.     

Helicobacter pylori cagA Status and s and m Alleles of vacA in Isolates from Individuals with a Variety of H. pylori-Associated Gastric Diseases

The cagA gene was detected in 100% of 16 Helicobacter pylori isolates from patients with gastric carcinoma versus 78% of 18 isolates from patients with duodenal ulcers (P = 0.344) and only 64% of 22 isolates from patients with gastritis only (P = 0.005) in Brazil. Also, there was a significant association between isolation of cagA⁺ s1-type vacA H. pylori in cases of stomach cancer and ulcers as opposed to cases of gastritis only (P = 0.004), but this was not true in Houston (P = 0.238), where 94% of all isolates were cagA⁺.     

Pediatric Helicobacter pylori Isolates Display Distinct Gene Coding Capacities and Virulence Gene Marker Profiles

Helicobacter pylori strains display remarkable genetic diversity, and the presence of strains bearing the toxigenic vacA s1 allele, a complete cag pathogenicity island (PAI), cagA alleles containing multiple EPIYA phosphorylation sites, and expressing the BabA adhesin correlates with development of gastroduodenal disease in adults. To better understand the genetic variability present among pediatric strains and its relationship to disease, we characterized H. pylori strains infecting 47 pediatric North American patients. Prevalence of mixed infection was assessed by random amplified polymorphic DNA analysis of multiple H. pylori clones from each patient. Microarray-based comparative genomic hybridization was used to examine the genomic content of the pediatric strains. The cagA and vacA alleles were further characterized by allele-specific PCR. A range of EPIYA motif configurations were observed for the cagA gene, which was present in strains from 22 patients (47%), but only 19 (41%) patients contained a complete cag PAI. Thirty patients (64%) were infected with a strain having the vacA s1 allele, and 28 patients (60%) had the babA gene. The presence of a functional cag PAI was correlated with ulcer disease (P = 0.0095). In spite of declining rates of H. pylori infection in North America, at least 11% of patients had mixed infection. Pediatric strains differ in their spectrum of strain-variable genes and percentage of absent genes in comparison to adult strains. Most children were infected with H. pylori strains lacking the cag PAI, but the presence of a complete cag PAI, in contrast to other virulence markers, was associated with more severe gastroduodenal disease.It is estimated that >50% of the world''s population is colonized with Helicobacter pylori in the stomach, making it one of the most common bacterial pathogens of humans. H. pylori infection is generally acquired in childhood (24, 33) and can persist for life. Gastritis (inflammation of the gastric mucosa) results in all who are colonized with H. pylori, but some hosts remain asymptomatic, while others develop peptic ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphoma. Gastric cancer is the second leading cause of cancer death worldwide, and 63% of gastric cancer cases in 2002 were attributable to H. pylori infection (38, 49). While severe disease most often presents in adulthood, children display H. pylori-associated gastritis and the incidence of ulcer disease among infected children was 6.8% in a European pediatric population (31). Many studies have examined bacterial, host, and environmental risk factors associated with development of H. pylori-associated diseases in adults, but similar studies in children have been limited.Genetic differences among H. pylori strains contribute to differences in disease outcome among infected individuals in adult populations. The gene encoding VacA, which induces vacuolation of host cells, is present in nearly all H. pylori strains, but a number of allele types have been defined. Strains having the type s1 vacA signal sequence and the m1 vacA middle region allele (vacA s1/m1) are associated with ulcer disease (9). The cag pathogenicity island (PAI) encodes a type IV secretion system (T4SS) (1, 15) that translocates the CagA protein effector, also encoded in the island, into host cells. Presence of the cag PAI is associated with increased inflammation, promoting host cell interleukin-8 (IL-8) production, and cagA-positive strains are associated with peptic ulcers (50) as well as gastric cancer (13). Inside the host cell, CagA protein becomes tyrosine phosphorylated at C-terminal EPIYA (Glu-Pro-Ile-Tyr-Ala) sites by src family kinases, deregulates SHP-2, and induces the hummingbird phenotype (26, 45). Strains having more C-type EPIYA motifs, the major phosphorylation site, induce stronger effects on host cells and are associated with gastric cancer (7, 12, 35). The presence of a functional allele of babA, a gene encoding an adhesin that mediates binding to Lewis B antigens expressed on gastric epithelial cells, is associated with duodenal ulcer and gastric adenocarcinoma (21).While these H. pylori genes and alleles have been associated with disease outcome in adults, studies in children have provided mixed results. A recent study identified two genes (jhp0562, coding for a putative glycosyltransferase, and jhp0870, coding for an outer membrane protein) associated with peptic ulcer disease in children, but not adults, suggesting a different spectrum of genetic risk factors in adults and children (37). Studies using a whole-genome microarray-based approach have been done to investigate the variability in genomic content of H. pylori strains, but these studies have included mostly strains from adult patients (25, 29, 41, 42). Studies of the genetic variability of pediatric H. pylori strains have largely been limited to genes previously associated with virulence in adult populations. To better understand the genetic variability present among pediatric strains, we used whole-genome microarray-based comparative genomic hybridization to examine the genomic content of H. pylori strains isolated from symptomatic North American children and compared the pediatric isolate genetic variability to that observed in adult strains. We then examined the frequency of known virulence genes and virulence alleles among the pediatric H. pylori strains and the associations of strain genotype with the clinical and histological characteristics of the patients.     

Study of the oipA genetic diversity and EPIYA motif patterns in cagA-positive Helicobacter pylori strains from Venezuelan patients with chronic gastritis

CagA and OipA are involved, among other virulence factors, in the ability of Helicobacter pylori to colonize the gastric mucosa and to modulate the host environment during the establishment of chronic infection. The number and type of EPIYA phosphorylation motifs and the presence and functional status of oipA have been involved in the induction of cellular transformations playing an important role in the development of H. pylori associated gastric diseases. This work determined the prevalence of the oipA virulence factor and EPIYA motif patterns in cagA-positive H. pylori gastric biopsies from chronic gastritis patients from the Central-Western region of Venezuela. DNA was extracted directly from gastric biopsies collected by upper endoscopy from 113 patients. The EPIYA motif genotyping and oipA gene functional status was determined by PCR and sequencing. Phylogenetic analysis with the 3′ variable region of cagA sequences was performed. Only Western-type EPIYA variants were detected: ABC (68.14%), ABCC (29.20%) and ABCCC (2.66%). High prevalence of strains with the oipA gene (93.8%) and its functional status “ON” (83%) was observed. No significant association between EPIYA motif patterns or oipA functional status with the histological changes in the gastric mucosa was found. Our study demonstrated the absolute predominance of the Western-type cagA gene in a Venezuelan admixed population. This is the first report showing oipA status of H. pylori strains in Venezuela. Further studies with a larger number of samples and including other pathologies are necessary to continue evaluating the role of the H. pylori virulence factors in the prevalence of gastric diseases in our country.     

Infection with<Emphasis Type="Italic"> cagA</Emphasis>-Positive and<Emphasis Type="Italic"> cagA</Emphasis>-Negative Types of<Emphasis Type="Italic"> Helicobacter pylori</Emphasis> Among Children and Adolescents with Gastrointestinal Symptoms in Latvia

In order to determine the prevalence of concomitant cagA-positive and cagA-negative Helicobacter pylori genotypes in individual subjects, a group of 56 symptomatic patients (aged 8–18 years) was studied. Among 31 patients culture-positive for Helicobacter pylori, only cagA-positive colonies were isolated from 18 patients, both cagA-positive and cagA-negative genotypes were isolated from 4 patients, and in 9 patients all of the individual colonies isolated were cagA-negative, but in seven of them a pool of colonies was positive for cagA. Thus, the presence of both cagA-positive and cagA-negative genotypes in the same individual was identified in 11 of the 31 culture-positive patients tested, and most of the patients predominantly colonized by cagA-negative strains also harbored a small amount of cagA-positive strains. Previous or current infection with cagA-positive strains of Helicobacter pylori was observed in 50 of the 56 patients studied.     

High Prevalence of Clarithromycin Resistance and cagA,vacA, iceA2, and babA2 Genotypes of Helicobacter pylori in Brazilian Children

We isolated 45 Helicobacter pylori strains from 217 child patients. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was detected in 27%, 13%, 4%, and 0% of strains, respectively. The A2143G mutation was the most prevalent (67%) among clarithromycin-resistant strains. In addition, strain genotyping revealed a significant association between gastritis severity and the simultaneous presence of cagA, vacA s1m1, iceA2, and babA2 genes.Helicobacter pylori infection is found worldwide and constitutes a public health concern in many countries. Previous epidemiological studies have shown a high prevalence of H. pylori infection in Brazil (2, 20, 24). H. pylori infection, generally acquired in childhood, persists asymptomatically for decades in most individuals.Amoxicillin, tetracycline, metronidazole, and clarithromycin are frequently used, combined with proton pump inhibitors or bismuth salts, for the treatment of H. pylori infections (25). However, antibiotic resistance is frequently associated with eradication failure (3, 16). Resistance to metronidazole and clarithromycin is population dependent, and several studies suggest that clarithromycin resistance is higher in strains isolated from children than in strains isolated from adults (10). In Brazil, the prevalence of clarithromycin-resistant strains in adults is reported to be from 7 to 10% (15, 18). However, little is known about the prevalence of clarithromycin-resistant H. pylori infection in Brazilian children.The primary aims of this study were to determine the prevalence of clarithromycin-resistant H. pylori strains in children, to identify those isolates via rapid methodology, and to examine the severity of gastritis caused by the antibiotic-resistant H. pylori isolates. Metronidazole, amoxicillin, and tetracycline resistance was also studied. Furthermore, the study aimed to genotype the vacA and iceA genes and to detect the cagA gene in gastric biopsy specimens, since recent studies found a high frequency of cagA-positive and iceA2-positive strains as well as a strain with the vacA signal region genotype s1 and middle region sequence m1 among pediatric H. pylori isolates in Brazil (6, 7, 11, 23). This is also the first investigation of babA2 gene prevalence in Brazilian children.A total of 217 consecutive child patients, aged 1 to 18 years (mean age, 10 years) (105 girls and 112 boys), who underwent upper gastrointestinal endoscopy for the evaluation of dyspeptic symptoms at the outpatient clinic of Pediatric Gastroenterology at the Instituto da Criança, Faculdade de Medicina da Universidade de São Paulo, during 2008 and 2009 were included. The study was approved by the Ethics Committee of the University Hospital. Patients previously treated for H. pylori infections were not included.Gastric biopsy specimens were processed for histological examination and evaluated according to the updated Sydney system of classification and grading of gastritis (4).Antral gastric specimens were transported in sodium thioglycolate broth (Difco, Detroit, MI) in an ice bath and ground before submission to DNA extraction and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with primers specific to the H. pylori 23S rRNA gene (17). The QIAmp tissue kit (Qiagen) was used for DNA extraction. Point mutations related to clarithromycin resistance in the 23S rRNA amplicon were investigated in all H. pylori isolates by PCR-RFLP using BsaI and MboII enzymes (27). The vacA, cagA, iceA, and babA2 genotypes were detected by PCR, as described elsewhere (1, 9, 21, 26, 28). In each experiment, H. pylori strain 26695 (ATCC 700392) was used as the positive-control strain.H. pylori strains were cultured on Belo Horizonte medium (22) under microaerophilic atmosphere at 37°C for 3 to 7 days, and the isolates were identified by Gram staining and biochemical tests for oxidase, catalase, and urease production. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was determined by the disc diffusion method (Oxoid), and MICs were determined by the Etest according to the manufacturer''s recommendations (AB Biodisk, Solna, Sweden). An isolate was considered resistant to clarithromycin or tetracycline if the MIC was >1 mg/liter and to metronidazole or amoxicillin if the MIC was >4 mg/liter (19).Data were analyzed by the two-tailed χ² test and Fisher exact test. P values of <0.05 were considered statistically significant.H. pylori was isolated in 45 (20.7%) of the 217 children; 12 (26.7%) of the 45 strains were clarithromycin resistant, 6 (13.3%) were metronidazole resistant, and 2 (4.4%) were amoxicillin resistant. All cultured H. pylori strains were susceptible to tetracycline (Fig. ​(Fig.1).1). No histological differences were observed between biopsy specimens with antibiotic-resistant strains and those with susceptible strains. PCR-RFLP was performed with all 12 clarithromycin-resistant isolates: 8 had the 23S rRNA A2143G point mutation, and 4 had the 23S rRNA A2142G mutation.Open in a separate windowFIG. 1.Distribution of MICs for the 45 H. pylori strains.Among the 45 H. pylori-infected children, 13 had mild chronic gastritis, 28 had moderate chronic gastritis, 2 had marked chronic gastritis, and 2 had normal gastric mucosa. The percentage of H. pylori-infected children with chronic gastritis was 95.5% (43 patients), while 4.4% of the children (2 patients) had normal mucosa (P < 0.001).vacA was detected in all 45 H. pylori-positive gastric biopsy specimens. The vacA genotypes s1m1, s2m2, and s1m2 or s2m1 were found in 57.7, 33.3, and 4.4% of the specimens, respectively. The iceA1 allele was detected in 9 (20%) and the iceA2 allele in 31 (68.9%) of the samples. Of the 45 H. pylori-positive biopsy specimens, 28 (62%) were cagA positive and 38 (84.4%) were babA2 positive. Correlation of histopathology results with vacA, cagA, and iceA genotypes showed that vacA s1m1-, cagA-, and iceA2-positive strains were more frequently found in patients with moderate and marked gastritis (77%) than in patients with mild gastritis (23%) (P < 0.001). Interestingly, in Slovenian children, vacA s1 and cagA were also shown to be associated with more pronounced chronic gastritis (12). In contrast, in Korean children, although vacA s1m1 cagA iceA1 was the predominant genotype, no association with gastritis severity was observed (14).In conclusion, we found a high incidence of clarithromycin-resistant H. pylori strains (27%) in Brazilian children. Furthermore, we found an association between clarithromycin resistance and either the vacA s1m1 (P = 0.007) or the iceA2 (P = 0.038) genotype. The high level of clarithromycin resistance among strains from children compared to adults (15, 18) suggests the importance of susceptibility testing, especially in Brazilian children. All together, these data stress the relevance of susceptibility testing and genotyping for establishing antibiotic treatment in pediatric H. pylori infection.In our study, PCR-RFLP proved to be a rapid and accurate method for the detection of clarithromycin resistance gene mutation directly in gastric biopsy samples. Only a few groups have studied mutations involved in clarithromycin resistance in strains obtained from children, and their results are similar to those obtained in our study (5, 13, 29).Our data also demonstrate an association between H. pylori infection and gastritis in Brazilian children. In addition, we confirmed the reported association of infection with vacA s1m1 cagA iceA2-positive H. pylori strains and gastritis severity (6, 11, 23). Furthermore, a high frequency of babA2 was found among H. pylori isolates. Previous studies of adults in Brazil reported a high prevalence of babA2-positive strains from patients with different upper gastrointestinal diseases (8). The high incidence of babA2 in H. pylori Brazilian isolates suggests that this gene could be a useful marker for identifying patients with a high risk of H. pylori infection in Brazil.     

Profile of Helicobacter pylori cagA & vacA genotypes and its association with the spectrum of gastroduodenal disease

PurposeGlobally, H. pylori virulence factors cagA and vacA genotypes and its variation is leading to the austere form of the gastroduodenal disease. Our objectives were to detect H. pylori in dyspeptic patients from biopsy samples with the validation of the various existing diagnostic tools and to screen the cagA, vacA genotypes profile from biopsy specimens and how it impacts in progression of gastroduodenal disease in southern India.Methods374 patients who attended endoscopy unit at Kasturba Hospital, Manipal with their consent obtained their biopsies. H. pylori were detected by HPE, Culture, RUT and PCR and its virulence gene were patterned with PCR.ResultsThe positive rate of H. pylori by HPE, RUT, Culture and PCR were 51.33%, 47.1%, 32.4% and 50.3% respectively and comparison by Bayesian LCMs analysis showed PCR is superior among them. The frequency of H. pylori virulence gene viz cagPAI (cagA) were 80.9%, and vacA alleles-s1m1 (42%), s1m2 (33%) and s2m2 (25%) genotypes by PCR respectively. Four combinations of cagA/vacA genotypes were noted, majority of strains harboured cagA⁺/vacA s1m1 genotypes (42.6%), interestingly this hyper-virulent strain more frequently seen in severe gastroduodenal disease whereas cagPAI negative strains as well as cagA⁻/vacA s2m2 combinations (19.1%) are seen most commonly in functional dyspepsia cases and depicted significant association by Chi-square test.ConclusionsThis study validates and compares the existing diagnostic methods for detecting H. pylori in biopsies. Also, it reveals some pattern of virulence gene combination will play a vital role in disease progression.