Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases

The vacuolating cytotoxin and the cytotoxin-associated protein, encoded by vacA and cagA, respectively, are important virulence determinants of Helicobacter pylori. Sixty-five H. pylori strains were isolated from dyspeptic patients (19 with peptic ulcer disease, 43 with chronic gastritis, and 3 with gastric cancer) and studied for differences in the vacA and cagA genes and their relationship to VacA and CagA expression, cytotoxin activity, and the clinical outcome of infection. By PCR, fifty-four (83.1%) of 65 strains had the vacA signal sequence genotype s1 and only 10 (15.4%) had the type s2. After primer modification, the vacA middle-region types m1 and m2 were detected in 24 (36.9%) and 41 (63.1%) strains, respectively. The combinations s1-m2 (31 [47.7%]) and s1-m1 (23 [35.4%]) occurred more frequently than s2-m2 (10 [15.4%]) (P = 0.01). No strain with the combination s2-m1 was found. All 19 patients with peptic ulcers harbored type s1 strains, in contrast to 32 (74.4%) of 43 patients with gastritis (P = 0.02). The vacA genotype s1 was associated with the presence of cagA (P < 0.0001), VacA expression (P < 0.0001), and cytotoxin activity (P = 0.003). The cagA gene was detectable in 48 (73.8%) of 65 isolates and present in 16 (84.2%) of 19 ulcer patients and 29 (67.4%) of 43 patients with gastritis (P = 0.17). The vacA genotypes of German H. pylori isolates are identical to those previously reported. H. pylori strains of vacA type s1 are associated with the occurrence of peptic ulceration and the presence of cagA, cytotoxin activity, and VacA expression.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Differences in Genome Content among Helicobacter pylori Isolates from Patients with Gastritis,Duodenal Ulcer,or Gastric Cancer Reveal Novel Disease-Associated Genes

Helicobacter pylori establishes a chronic infection in the human stomach, causing gastritis, peptic ulcer, or gastric cancer, and more severe diseases are associated with virulence genes such as the cag pathogenicity island (PAI). The aim of this work was to study gene content differences among H. pylori strains isolated from patients with different gastroduodenal diseases in a Mexican-Mestizo patient population. H. pylori isolates from 10 patients with nonatrophic gastritis, 10 patients with duodenal ulcer, and 9 patients with gastric cancer were studied. Multiple isolates from the same patient were analyzed by randomly amplified polymorphic DNA analysis, and strains with unique patterns were tested using whole-genome microarray-based comparative genomic hybridization (aCGH). We studied 42 isolates and found 1,319 genes present in all isolates, while 341 (20.5%) were variable genes. Among the variable genes, 127 (37%) were distributed within plasticity zones (PZs). The overall number of variable genes present in a given isolate was significantly lower for gastric cancer isolates. Thirty genes were significantly associated with nonatrophic gastritis, duodenal ulcer, or gastric cancer, 14 (46.6%) of which were within PZs and the cag PAI. Two genes (HP0674 and JHP0940) were absent in all gastric cancer isolates. Many of the disease-associated genes outside the PZs formed clusters, and some of these genes are regulated in response to acid or other environmental conditions. Validation of candidate genes identified by aCGH in a second patient cohort allowed the identification of novel H. pylori genes associated with gastric cancer or duodenal ulcer. These disease-associated genes may serve as biomarkers of the risk for severe gastroduodenal diseases.Infection with Helicobacter pylori is one of the most common bacterial infections in humans worldwide, and like the case for many other infections, rates are higher in developing countries (80 to 90%) than in developed countries (<50%) (20, 33, 39). The infection is associated with peptic ulcers, gastric carcinoma, and mucosa-associated lymphoma (32, 36, 39). Most infected people remain asymptomatic during their lifetime, and only about 15% develop gastroduodenal illness. Environmental, host, and bacterial factors all play a role in the outcome of the infection. A number of bacterial virulence factors associated with disease have been described for H. pylori, and the most consistently reported are the cag pathogenicity island (cag PAI) (4, 40, 52) and vacA (2, 43). Outer membrane proteins such as BabA2 (18), OipA (54), and SabB (11), as well as the iceA gene, have also been reported to be associated with disease. An outstanding characteristic of H. pylori is the high level of genetic diversity among isolates from different patients, even if they belong to the same ethnic population. Several molecular typing techniques have been used to genotype H. pylori strains from different populations, demonstrating genetic differences among populations and even among isolates from the same individual, suggesting the presence of mixed infection (3, 9, 19, 26, 35, 46). The ability of this bacterium to generate such genetic diversity is due to its natural competence, high recombination and mutation rates, or the occurrence of slipped-strand synthesis and phase variation (17, 24).H. pylori was the first bacterial species for which whole genome sequences of two independent strains were available (J99 and 26695). Their comparison showed that approximately 6 to 7% of the H. pylori genes present in one strain are absent in the other. These genes are called strain-specific genes, and almost half of them are located in hypervariable regions of the genome (1, 51). These regions contain a considerable number of restriction-modification genes and genes for transposases, topoisomerases, and outer membrane proteins. One of these regions is the cag PAI, whereas the others have been termed plasticity zones (PZs). Whole-genome DNA microarrays facilitated further analyses of the genomic contents of 15 H. pylori clinical isolates, revealing 362 genes (22% of all genes) that are not conserved among strains and represent variable or strain-specific genes (45). Similar microarray-based comparative genomic hybridization (aCGH) studies have been used to explore the genetic diversity in the H. pylori strain population colonizing the different regions of the stomach of a single host for both adults and children (46) and to correlate the genetic contents of H. pylori strains with pathogenesis in animal models (6, 25). These studies indicate that H. pylori strains gain or lose loci during chronic infection, suggesting a continuous genetic flux, mainly inside the PZs (14, 22, 26, 27, 28). The sequence of an H. pylori strain isolated from a patient with chronic atrophic gastritis was recently published, identifying additional strain-specific genes (38) and, by comparison with previous studies (22), suggesting that 121 genes are “chronic atrophic gastritis associated.”Other studies have reported that genes located in the PZs, such as jhp0947 and jhp0949, are associated with disease (12, 37, 47). The dupA gene was associated with an increased risk for duodenal ulcer (DU) and a low risk for gastric atrophy and cancer (31). The aim of the present study was to compare the genomic contents of H. pylori strains isolated from patients with nonatrophic gastritis (NAG), DU, or gastric cancer (GC) in a Mexican-Mestizo population in order to look for genes previously associated with severe gastroduodenal diseases and to identify novel disease biomarkers.     

homB Status of Helicobacter pylori as a Novel Marker To Distinguish Gastric Cancer from Duodenal Ulcer

The hom family of Helicobacter pylori outer-membrane proteins, especially the homB gene, has been suggested as a novel virulence factor; however, the clinical association and function of this gene are still unclear. We evaluated the presence of the homA, homB, and cagA genes in 286 strains isolated from patients in the U.S. and Colombian populations (126 with gastritis, 96 with duodenal ulcer, and 64 with gastric cancer) by PCR. The results were compared with the clinical presentation and gastric injury. The prevalence of the homB gene was significantly higher in strains isolated from gastric-cancer patients (71.9%) than in those from duodenal ulcer patients (52.1%) (P = 0.012). In a multivariate analysis, the presence of the cagA gene significantly increased the risk for developing gastric cancer and duodenal ulcer, with the presence of the homB gene acting as a factor that could distinguish gastric cancer from duodenal ulcer (adjusted odds ratio, 3.033; 95% confidence interval, ∼1.37 to ∼6.73). cagA status was correlated with homB status (r = 0.323; P < 0.01). A histological analysis showed that cagA status was associated with inflammation and atrophy both in the antrum and in the corpus, while homB status was associated with inflammation and atrophy in the corpus. homB gene status might be susceptible to gastric-cancer development such that the homB gene is used as a factor for discriminating the risk of gastric cancer from that of duodenal ulcer.Helicobacter pylori infection is one of the most common infections of mankind and is etiologically associated with gastritis, peptic ulcer disease (PUD), gastric cancer (GC), and gastric mucosa-associated lymphoid tissue lymphoma (19). Most infected people remain asymptomatic. Factors thought to be associated with clinical gastroduodenal diseases include H. pylori virulence, host genetics, and environmental factors, such as diet (11). Putative H. pylori virulence factors associated with an increased risk of a clinical outcome include the cag pathogenicity island, CagA, VacA, BabA, and OipA (5, 9, 22). However, none have been exclusively linked to a specific H. pylori-related disease (e.g., GC).The hom family is a small paralogous family of proteins that contain the C-terminal alternating hydrophobic motif and signal sequences typical of outer-membrane proteins. The homB and homA genes are 90% identical; the differences are confined to the central domain (1). Recent studies suggested that there was a close association between the presence of the homB gene and interleukin-8 secretion from human gastric epithelial cells and that the number of H. pylori isolates binding to gastric cells was related to the number of homB copies present (15). Moreover, the authors proposed that the presence of the homB gene was significantly associated with PUD in Portuguese children and adults less than 40 years of age and that it may be a new H. pylori virulence factor (15, 16). However, there is no study for the association between the homB gene and H. pylori-related diseases in other countries. This study investigated whether there was an association between the homA and homB genes and clinical gastroduodenal diseases and the severity of gastric inflammation in the U.S. and Colombian populations.     

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.     

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.     

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.     

Helicobacter pylori Type I Strains Among Austrian and Portuguese Patients with Gastritis, Peptic Ulcer or Gastric Cancer

 The frequency of occurrence of Helicobacter pylori type I strains in isolates from Austria and Portugal and different polymerase chain reaction-based approaches to detect the cag pathogenicity island were assessed. Of the 41 Austrian strains, eight of 14 (57.2%) isolated from patients with gastritis, 14 of 19 (73.7%) from patients with peptic ulcer and eight of eight (100%) from patients with gastric cancer were type I strains. Among the Portuguese strains, eight of 14 (57.2%) isolated from patients with gastritis, ten of 12 (83.3%) from patients with peptic ulcer and five of 13 (38.5%) from patients with gastric cancer were classified as type I. Thus, Helicobacter pylori type I strains occur frequently in both populations but show no significant correlation with peptic ulcer disease. The prevalence of the type I genotype in Austrian cancer patients, however, was significantly higher (P=0.007). The cagE-specific polymerase chain reaction was found to be a reliable and efficient method for detection of the cag pathogenicity island.     

Role of Interleukin-32 in Helicobacter pylori-Induced Gastric Inflammation

Helicobacter pylori infection is associated with gastritis and gastric cancer. An H. pylori virulence factor, the cag pathogenicity island (PAI), is related to host cell cytokine induction and gastric inflammation. Since elucidation of the mechanisms of inflammation is important for therapy, the associations between cytokines and inflammatory diseases have been investigated vigorously. Levels of interleukin-32 (IL-32), a recently described inflammatory cytokine, are increased in various inflammatory diseases, such as rheumatoid arthritis and Crohn''s disease, and in malignancies, including gastric cancer. In this report, we examined IL-32 expression in human gastric disease. We also investigated the function of IL-32 in activation of the inflammatory cytokines in gastritis. IL-32 expression paralleled human gastric tissue pathology, with low IL-32 expression in H. pylori-uninfected gastric mucosa and higher expression levels in gastritis and gastric cancer tissues. H. pylori infection increased IL-32 expression in human gastric epithelial cell lines. H. pylori-induced IL-32 expression was dependent on the bacterial cagPAI genes and on activation of nuclear factor κB (NF-κB). IL-32 expression induced by H. pylori was not detected in the supernatant of AGS cells but was found in the cytosol. Expression of the H. pylori-induced cytokines CXCL1, CXCL2, and IL-8 was decreased in IL-32-knockdown AGS cell lines compared to a control AGS cell line. We also found that NF-κB activation was decreased in H. pylori-infected IL-32-knockdown cells. These results suggest that IL-32 has important functions in the regulation of cytokine expression in H. pylori-infected gastric mucosa.     

Genes Required for Assembly of Pili Associated with the Helicobacter pylori cag Type IV Secretion System

Helicobacter pylori causes numerous alterations in gastric epithelial cells through processes that are dependent on activity of the cag type IV secretion system (T4SS). Filamentous structures termed “pili” have been visualized at the interface between H. pylori and gastric epithelial cells, and previous studies suggested that pilus formation is dependent on the presence of the cag pathogenicity island (PAI). Thus far, there has been relatively little effort to identify specific genes that are required for pilus formation, and the role of pili in T4SS function is unclear. In this study, we selected 7 genes in the cag PAI that are known to be required for T4SS function and investigated whether these genes were required for pilus formation. cagT, cagX, cagV, cagM, and cag3 mutants were defective in both T4SS function and pilus formation; complemented mutants regained T4SS function and the capacity for pilus formation. cagY and cagC mutants were defective in T4SS function but retained the capacity for pilus formation. These results define a set of cag PAI genes that are required for both pilus biogenesis and T4SS function and reveal that these processes can be uncoupled in specific mutant strains.     

Chemokines and Antimicrobial Peptides Have a cag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.     

High Prevalence of Clarithromycin-Resistant Helicobacter pylori Strains and Risk Factors Associated with Resistance in Madrid,Spain

Clarithromycin is one of the antibiotics used for the treatment of Helicobacter pylori infections, and clarithromycin resistance is the most important factor when it comes to predicting eradication failure. The present study analyzed H. pylori isolates for the presence of 23S rRNA gene mutations and determined the risk factors associated with resistance among H. pylori isolates collected in Madrid, Spain, in 2008. We studied 118 H. pylori strains isolated from the same number of patients. A total of 76.3% of the patients were born in Spain, 52.7% were children, 20.3% had previously been treated, and 66.1% were female. Clarithromycin resistance was determined by Etest. H. pylori strains were considered resistant if the MIC was ≥1 mg/liter. DNA extraction was carried out by use of the NucliSens easyMAG platform with NucliSens magnetic extraction reagents (bioMérieux). The DNA sequences of the 23S rRNA genes of clarithromycin-resistant and -sensitive strains were determined to identify specific point mutations. The vacA genotype and cagA status were determined by PCR. We found that 42 (35.6%) strains were resistant to clarithromycin by Etest. Etest results were confirmed by detection of the presence of point mutations in 34 (88.1%) of these strains. Eight H. pylori strains were resistant to clarithromycin by Etest but did not have a point mutation in the 23S rRNA gene. Mutation at A2143G was found in 85.3% of the strains, mutation at A2142G in 8.8%, and mutation at T2182C in 5.9%. Dual mutations were found in 8.8% of the strains. H. pylori clarithromycin-resistant strains were strongly associated with pediatric patients, with patients born in Spain, and with patients who had previously been treated (P ≤ 0.02). In addition, H. pylori strains resistant to clarithromycin more frequently presented the vacA s2/m2 genotype and were more likely to be cagA negative than susceptible strains (39.1% and 11.2%, respectively; P value < 0.001). We concluded that, in the present study, H. pylori clarithromycin-resistant strains are more frequently found in children, in patients mostly born in Spain, and in individuals who were previously treated for H. pylori infection and that these individuals are more likely colonized with a less virulent H. pylori strain.Helicobacter pylori is a microaerobic, Gram-negative spiral bacterium that colonizes the human stomach and is found in more than half of the world''s population (32). Infections with H. pylori are closely associated with chronic gastritis, peptic ulcer disease, and the development of gastric cancer (8, 32).All consensus guidelines recommend eradication of H. pylori for patients with symptoms (9, 28). Standard therapy combines a proton pump inhibitor (PPI) or ranitidine bismuth citrate and two antibiotics, chosen from among amoxicillin, clarithromycin, and metronidazole (24, 25). However, this therapy has been questioned because of the increased eradication failure rates. Many factors have been implicated as causes of treatment failure, including ineffective penetration of antibiotics into the gastric mucosa, antibiotic inactivation by the low stomach pH, a lack of patient compliance, and the emergence of acquired resistance to antibiotics by H. pylori (26, 27).In many cases, clarithromycin is the key component of these combination therapies. However, resistance to clarithromycin has become one of the major reasons for treatment failure (13). The prevalence of H. pylori resistance to clarithromycin varies among different countries, such as 10.6% to 25% in North America, 16% in Japan, and 1.7% to 23.4% in Europe (14, 19, 21). Overall, resistance to clarithromycin has been detected more in patients living in the south than in those living in the north of Europe (21). Fewer studies have focused on the prevalence of clarithromycin resistance among H. pylori strains from children compared with that among strains from the adult population. These studies will be useful for estimating the rate of clarithromycin-resistant H. pylori isolates among children and adults in Spain in the future (1, 23).Clarithromycin acts by binding to the peptidyltransferase region of 23S rRNA and inhibits protein synthesis (36). The resistance to clarithromycin in H. pylori has been shown to be due to point mutations in the peptidyltransferase region of domain V of the 23S rRNA. Two copies of the 23S rRNA gene are present in H. pylori, and the most common mutation is an A-to-G transition at position 2143 (A2143G) (13, 36), but several point mutations, at positions A2142G, A2144G, and T2182C, have been described. Recent reports have indicated that other mutations, such as A2115G, G2141A, C2147G, T2190C, C2195T, A2223G, and C2694A, might also be associated with clarithromycin resistance (20, 31). Other mechanisms of resistance, such as methylase production, the actions of macrolide-inactivating enzymes, and active efflux, have been described in several bacteria. Active efflux has also recently been described in H. pylori (22).Since the worldwide increase in the rate of clarithromycin resistance represents a problem of relevance, some studies have been performed in order to identify its relationship with bacterial genetic factors (12, 35, 38).Two genes (cytotoxin-associated gene A [cagA] and vacuolation-associated gene A [vacA]) have been identified to be the main virulence factors. cagA is located in the cag pathogenicity island (PAI), which encodes a type IV secretion system, and the presence of cagA is closely associated with more severe gastric diseases (2, 15, 34). The VacA toxin induces vacuole formation in the host cells. There is considerable variation in vacuolation activity among H. pylori strains, primarily due to differences in the vacA gene structure in the signal region (s1 and s2) and the middle region (m1 and m2). vacA s1/m1 and s1/m2 produce high and moderate levels of VacA toxin, respectively, whereas s2/m2 produces little or no toxin (11). A strong association between clarithromycin susceptibility and these virulence factors has been reported (12, 38).The focus of the present study was to evaluate the distribution of clarithromycin-resistant H. pylori strains and their association with genotypic markers, such as the cagA gene and allelic variants of the vac gene. We also examined the distribution of H. pylori clarithromycin resistance in relation to the patient''s age, place of birth, and history of treatment. Our main goal is to determine potential host and bacterial factors that may help in predicting resistance to clarithromycin among H. pylori isolates.     

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.