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.     

Analysis of the 3′-variable region of the cagA gene from Helicobacter pylori strains infecting patients at New York City hospitals

Helicobacter pylori infects the gastric mucosa in humans and is a causative agent for peptic ulcer disease (PUD) and gastric cancer (GC). CagA is produced by H. pylori and is associated with more severe outcomes. cagA genes vary at the 3′-region with respect to phosphorylation motifs (EPIYA-A, -B, -C, or -D) and CagA multimerization motifs (CM). This variability may be associated with the clinical outcomes. We examined the variable region of cagA genes expressed in H. pylori-infected patients treated at three NYC Hospitals. DNA was isolated from gastric biopsies of patients undergoing upper endoscopy. Most H. pylori-infected patients were Black or Hispanic. The cagA 3′-region of CagA-positive samples was amplified by PCR, purified and sequenced. The patterns of EPIYA and CM motifs were examined and related to clinical outcomes. We obtained 42 CagA sequences from our sample collection. The EPIYA phosphorylation motif pattern was ABC in 81.0% of our samples. Western (W) and Eastern (E) CM motifs have also been defined. CagA proteins lacking an Eastern CM motif and possessing one or two Western CM motifs were observed more frequently in patients with PUD and GC when compared with non-ulcer gastritis (50.0% vs 11.8%, respectively), suggesting that these CM motif patterns are more virulent than those containing at least one Eastern CM motif. We conclude that In H. pylori-infected patients treated at NYC Hospitals, CM motif patterns in the CagA 30-variable region may be more significant than EPIYA motif patterns with respect to clinical outcomes.     

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.     

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.     

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.     

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.     

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.     

CagA C-terminal variations in Helicobacter pylori strains from Colombian patients with gastric precancerous lesions

The C-terminus of the Helicobacter pylori CagA protein is polymorphic, bearing different EPIYA sequences (EPIYA-A, B, C or D), and one or more CagA multimerization (CM) motifs. The number of EPIYA-C motifs is associated with precancerous lesions and gastric cancer (GC). The relationship between EPIYA, CM motifs and gastric lesions was examined in H. pylori-infected Colombian patients from areas of high and low risk for GC. Genomic DNA was extracted from H. pylori strains cultured from gastric biopsies from 80 adults with dyspeptic symptoms. Sixty-seven (83.8%) of 80 strains were cagA positive. The 3’ region of cagA was sequenced, and EPIYA and CM motifs were identified. CagA proteins contained one (64.2%), two (34.3%) or three EPIYA-C motifs (1.5%), all with Western type CagA-specific sequences. Strains with one EPIYA-C motif were associated with less severe gastric lesions (non-atrophic and multifocal atrophic gastritis), whereas strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinal metaplasia and dysplasia) (p <0.001). In 54 strains, the CM motifs were identical to those common in Western strains. Thirteen strains from the low-risk area contained two different CM motifs: one of Western type located within the EPIYA-C segment and another following the EPIYA-C segment and resembling the CM motif found in East Asian strains. These strains induced significantly shorter projections in AGS cells and an attenuated reduction in levels of CagA upon immunodepletion of SHP-2 than strains possessing Western/Western motifs. This novel finding may partially explain the difference in GC incidence in these populations.     

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.     

Local and Systemic Immune and Inflammatory Responses to Helicobacter pylori Strains

Colonization with Helicobacter pylori eventuates in varied clinical outcomes, which relate to both bacterial and host factors. Here we examine the relationships between cagA status, serum and gastric juice antibody responses, and gastric inflammation in dyspeptic patients. Serum, gastric juice, and gastric biopsy specimens were obtained from 89 patients undergoing endoscopy. H. pylori colonization and cagA status were determined by histology, culture, and PCR methods, and acute inflammation and chronic inflammation in the gastric mucosa were scored by a single pathologist. Serum and gastric juice antibodies to H. pylori whole-cell and CagA antigens were determined by enzyme-linked immunosorbent assay. Relationships between variables were sequentially analyzed using univariate and multivariate statistical methods. Of the 89 subjects, 62 were colonized by H. pylori. By univariate analyses, levels of serum immunoglobulin G (IgG) and IgA and gastric juice IgA antibodies against whole-cell and CagA antigens each were significantly higher in the H. pylori-positive group than in the H. pylori-negative group (P < 0.001). H. pylori and CagA seropositivities were both significantly associated with enhanced inflammation in gastric antrum and body (P < 0.02). The presence of gastric juice antibodies to H. pylori antigens was associated with more severe gastric inflammation. However, in multivariate analyses, only the presence of serum antibodies against CagA and, to a lesser extent, whole-cell antigens remained significantly associated with acute and chronic inflammation in antrum and body (P < 0.05). Thus, serum antibody response to CagA correlates with severity of gastric inflammation. Furthermore, given the relationships demonstrated by multivariate analysis, determination of gastric juice antibodies may provide a better representation of serum, rather than secretory, immune response.     

Characterization of the cagA-gene in Helicobacter pylori in Mongolia and detection of two EPIYA-A enriched CagA types

Helicobacter pylori infection is strongly associated with gastritis, gastroduodenal ulcer disease and gastric carcinoma. The virulence of H. pylori strains increases with the presence of the pathogenicity island PAI, which encodes a Type 4 Secretion System and the oncoprotein CagA. Two major CagA types can be distinguished by differences in the repetitive EPIYA region in the C-terminal sequence; the more virulent East Asian CagA type with EPIYA-A, -B, and -D motifs and the Western CagA type with EPIYA-A, -B, and C motifs, the virulence of which is associated with the multitude of EPIYA-C motifs.In this study, the cagA gene was characterized in H. pylori strains isolated from Mongolians suffering from gastritis (80%) or ulcer (20%). The EPIYA region of 53 isolates was determined by PCR-amplification of overlapping cagA regions and subsequent Sanger sequencing. Only one H. pylori isolate carried the East Asian type (ABD) and 52 isolates the Western type of CagA, thereof 30 the EPIYA type ABC, 19 the ABCC type and one each of type ABCCCC, AAABC and AAAAB. An amino acid exchange from EPIYA-B to EPIYT-B was predominantly found in CagA proteins in strains with < 2 EPIYA-C copies (n = 25/32; p = 0.015) including the two EPIYA-A enriched CagA proteins, which have not been described to date. Due to the amino acid triplet preceding the EPIYA motif and strength of predicted phosphorylation, the multiple EPIYA-A motifs A2, A3 and A4 were shown to cluster with EPIYA-B and EPIYT-B with the unique feature of amino acid E in position ? 4 to Y of EPIYA. It has been described that tyrosine-phosphorylated EPIYA-A and -B motifs counteract the EPIYA-C-driven signaling towards host cell transformation and malignancy. Thus, Mongolian H. pylori strains carrying CagA proteins not only with a few EPIYA-C segments but also with multiplied EPIYA-A segments are probably less virulent; a thesis that needs further investigation at the protein level.     

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.     

Detection of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in bioptates of gastric mucosa of patients with gastritis and gastric ulcers using real-time PCR

Helicobacter pylori is currently believed to play an important role in the pathogenesis of gastritis, gastric and duodenal ulcers, and stomach cancer. The ability of H. pylori to colonize gastric mucosa and cause inflammation is determined by genetic factors, three of which are the best studied: the pathogenicity factors CagA and VacA and the adhesion factor BabA. We developed primer systems and probes for real-time polymerase chain reaction (RT PCR), which allowed us to detect H. pylori in specimens and to perform typing of H. pylori virulence factors CagA, VacA, and BabA. Comparison of two H. pylori diagnostic techniques, histological studies of bioptates and RT PCR, demonstrated the superiority of RT PCR in specificity and sensitivity. Typing of H. pylori demonstrated that 70–80% of strains had the cagA⁺/vacA s1 genotype and 10–20% had the cagA/vacA s2 genotype; babA was detected in 75–85% of all strains. H. pylori typing data did not reveal any correlations between the pathogenicity factors CagA and VacA or the adhesion factor BabA with severity of infection.     

Diverse Characteristics of the cagA Gene of Helicobacter pylori Strains Collected from Patients from Southern Vietnam with Gastric Cancer and Peptic Ulcer

The pathogenesis of gastroduodenal diseases is related to the diversity of Helicobacter pylori strains. CagA-positive strains are more likely to cause gastric cancer than CagA-negative strains. Based on EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl terminus corresponding to phosphorylation sites, H. pylori CagA is divided into East Asian CagA and Western CagA. The former type prevails in East Asia and is more closely associated with gastric cancer. The present study used full sequences of the cagA gene and CagA protein of 22 H. pylori strains in gastric cancer and peptic ulcer patients from Southern Vietnam to make a comparison of genetic homology among Vietnamese strains and between them and other strains in East Asia. A phylogenetic tree was constructed based on full amino acid sequences of 22 Vietnamese strains in accordance with 54 references from around the world. The cagA gene was found in all Vietnamese H. pylori strains. Twenty-one of 22 (95.5%) strains belonged to the East Asian type and had similar characteristics of amino acid sequence at the carboxyl terminus to other strains from the East Asian region. From evidence of East Asian CagA and epidemiologic cancerous lesions in Vietnam, H. pylori-infected Vietnamese can be classified into a high-risk group for gastric cancer, but further studies on the interaction among environmental and virulence factors should be done. Finally, phylogenetic data support that there is a Japanese subtype in the Western CagA type.Helicobacter pylori is a gram-negative bacterium that infects about 50% of the world''s population. Infections with H. pylori can result in chronic active gastritis and are a risk factor for peptic ulcers, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (40, 52, 53, 56). The prevalence of H. pylori infections is not the same in different parts of the world. Recent studies reported that humans actually acquired H. pylori in the early days of their history, long before the migration of modern humans out of Africa, and the diverse distribution of H. pylori today is associated with waves of human migration in the past (19, 32, 36, 61, 62). The rate of H. pylori infections is high in Africa, East Asia, and South Asia; however, the incidence of gastric cancer is high in East Asia but not in South Asia or Africa; this may be explained partly by the diversity of H. pylori strains in these regions (62).In cases of gastroduodenal diseases, especially gastric cancer, the pathogenesis involves three major factors: H. pylori virulence factors, host factors, and environmental factors (1, 10, 14, 16, 21, 34, 57). Two H. pylori virulence factors that have been focused on in many studies all around the world are VacA and CagA. VacA is encoded by the vacA gene and found in all H. pylori strains. The vacA gene is classified into three major genotypes: s1/m1, s1/m2, and s2/m2. s1/m1 strains produce higher levels of cytotoxin than s1/m2 or s2/m2 strains (2, 3). CagA is encoded by cagA, located in the cagPAI (pathogenicity island) region of the H. pylori genome, and the presence of this protein is a marker of cagPAI. Unlike the vacA gene, the cagA gene has been found in only 50% to 70% of H. pylori strains infecting Western populations: however, 80% to 100% of H. pylori strains from East Asia have the cagA gene, with the cagPAI region, in their genome (17, 49, 55, 61, 64, 65). Patients infected with H. pylori strains possessing CagA were at greater risk of developing gastric adenocarcinoma than those uninfected or infected with CagA-negative strains (11, 41). CagA is injected directly from H. pylori into intragastric epithelial cells via the type IV secretion system. The injected CagA mimics eukaryotic adaptor proteins that recruit multiple host signaling factors into protein complexes that target cellular junctions, cell proliferation, and actin-cytoskeletal rearrangements (54). The translocated CagA undergoes tyrosine phosphorylation by Src and Abl family kinases and binds to SHP-2 in the human gastric mucosa (45, 47, 48). The repeated EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl-terminal end of CagA are the targets of tyrosine phosphorylation (8, 22, 24, 38, 63). CagA multimerization plays an important role in the pathophysiological activity of CagA in disturbing host cell functions via SHP-2 deregulation, and EPIYA polymorphisms of CagA greatly influence the magnitude and duration of phosphorylation-dependent CagA activity (37, 42). SHP-2, like its Drosophila melanogaster homolog Corkscrew, is known to play an important role in the mitogenic signal transduction that connects receptor tyrosine kinases and ras (20). It is possible that deregulation of SHP-2 by the translocation of CagA plays a role in the acquisition of a cellular-transformed phenotype at a relatively early stage in the carcinogenesis of gastric carcinoma. A recent study on generating CagA in transgenic mice has provided the first direct evidence of the role of CagA as a bacterium-derived oncoprotein that acts in mammals and further indicates the importance of tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms (39). Based on characteristics of the EPIYA motif, the H. pylori CagA protein could be divided into a Western type and an East Asian type. The East Asian CagA protein exhibits stronger SHP-2-binding activity and so is more pathogenic than the Western CagA protein in H. pylori-infected patients (4, 7, 23, 37). Clinical data from East Asia, Japan, and South Korea indicated that the East Asian form of CagA was more closely related to persistent active inflammation, atrophic gastritis, and a higher risk of gastric cancer than the Western form (5, 6, 26, 44). It is quite clear that by studying the characteristics of H. pylori strains, especially the cagA gene and CagA protein, one can categorize H. pylori-infected patients into those at high risk of developing gastric cancer and those not.Vietnam is a developing country located in Southeast Asia. However, according to historical and migrational evidence, the Vietnamese are more closely related to people from East Asia than people from South Asia. Gastric cancer is one of the five most common cancers in Vietnam, including lung, stomach, liver, recto-colon and naso-pharynx cancer in males. In females, it ranks third behind breast and cervical-uterine cancer. The prevalence of gastric cancer in Northern Vietnam is as high as that in China or Korea. Its prevalence in Southern Vietnam is lower but still higher than that in Thailand and South Asia (46, 50). A few studies reported that cagA was found in nearly 100% of H. pylori-infected Vietnamese (60, 61), but no studies have examined the type of CagA protein or the full sequence of cagA in Vietnamese patients. The present study reports the diverse characteristics of cagA and classification of CagA in H. pylori-infected patients from Southern Vietnam based on the full genomic cagA sequence.     

Vacuolating Cytotoxin Genotypes Are Strong Markers of Gastric Cancer and Duodenal Ulcer-Associated Helicobacter pylori Strains: a Matched Case-Control Study

The Helicobacter pylori virulence gene, cagA, and active forms of the vacuolating cytotoxin gene, vacA, are major determinants of pathogenesis. However, previous studies linking these factors to disease risk have often included patients using aspirin/nonsteroidal anti-inflammatory agents (NSAIDs) or acid-suppressing drugs, both of which may confound results. Also, particularly for gastric cancer (GC), controls have often been of quite different ages. Here, we performed a careful study in a “clean” Belgian population with gastric cancer cases age and sex matched to 4 controls and with a parallel duodenal ulcer (DU) group. As in other populations, there was a close association between the presence of cagA and the vacA s1 genotype. For GC, associations were found for vacA s1-positive (P = 0.01, odds ratio [OR], 9.37; 95% confidence interval [CI], 1.16 to 201.89), i1-positive (P = 0.003; OR, 12.08; 95% CI, 1.50 to 259.64), and cagA-positive status (P < 0.05; OR, infinity; 95% CI, 0.76 to infinity). For DU, associations were found with vacA s1 (P = 0.002; OR, 6.04; 95% CI, 1.52 to 27.87) and i1 (P = 0.004; OR, 4.35; 95% CI, 1.36 to 14.78) status but not with cagA status. Neither condition showed independent associations with the vacA m1 allele or with more biologically active forms of cagA with longer 3′ variable regions. In this Belgian population, the best markers of gastric cancer- and duodenal ulcer-associated strains are the vacA s1 and i1 genotypes. This fits with experimental data showing that the s and i regions are the key determinants of vacuolating cytotoxin activity.     

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.     

Antibody Response of Patients with Helicobacter pylori-Related Gastric Adenocarcinoma: Significance of Anti-CagA Antibodies

The aim of this study was to search for a specific antibody pattern in sera from patients suffering from Helicobacter pylori-related gastric adenocarcinoma (GAC). The serological response of 22 patients suffering from GAC, 31 patients with gastroduodenal ulcer, and 39 asymptomatic subjects was analyzed using immunoblotting performed with three H. pylori strains: strain ATCC 43579; strain B110, isolated from a patient with ulcers; and strain B225, isolated from a patient with GAC. In addition, the latex agglutination test Pyloriset Dry was used to analyze ambiguous sera. H. pylori seropositivity was 75% in the GAC group, 61.3% in the ulcer group, and 56.4% in the asymptomatic group. Anti-CagA antibodies were found more often in the GAC group (48.8%) and in the ulcer group (47.3%) than in the asymptomatic group (21.2%). These percentages depended on the strain used as an antigen: in the GAC group, the anti-CagA frequencies were 93.3, 40, and 13.3% with strains B225, B110, and ATCC 43579, respectively. Thus the presence of anti-CagA antibodies was increased in patients suffering from H. pylori-related GAC, in particular when the CagA antigen was from a GAC strain. These data suggest the existence of a CagA protein specifically expressed by H. pylori strains isolated from GAC patients.