Genotypes of Helicobacter pylori in Polish population

Here we have studied the genetic diversity of Helicobacter pylori strains recovered from 64 individual patients, 5 family members and 13 unsuccessfully treated patients. The recovered bacteria were finger-printed by the PCR-RFLP and RAPD methods and virulence associated loci (cagPAI, vacA) were PCR studied. Unique differentiation of every independently isolated strain from not-related persons was possible by RAPD technique. In PCR-RFLP technique several profile groups (7 and 15) for particular endonuclease tested were found. Eleven patients carried strains of the same gene profile (PCR-RFLP) and the same overall genotype (RAPD) before and after therapy. In the family studies, essentially the same strain was found in different relatives in three cases, and different strains were found in the other two cases. Island of cagPAI was present in 79% of all strains tested, half and one-fifth of all strains tested presented, s1am2 and s1m1 alleles of vacA gene, respectively. Independently from identity or diversity of pre- and post-treatment strains and strains recovered from the family members we have been observed identical cagPAI/vacA genotypes. These results suggest that H. pylori infections in Poland can be mixed, although just one strain may often predominate, and that inter-family transmission may be significant even in this high risk society. The genetic feature of virulence-associated loci are similar to those seen elsewhere in Europe, although strains that carry the cagPAI and the potentially more toxigenic alleles of the vacA gene are more common. RAPD technique is proven as most differentiating, however PCR-RFLP allows for easy recognition of mixed infection with two or more different strains. Molecular typing study in case of children therapy may allow reduce rate of relapses by reduction of possible transmission from family source.     

Molecular Evolution of the Helicobacter pylori Vacuolating Toxin Gene vacA

Helicobacter pylori is a genetically diverse organism that is adapted for colonization of the human stomach. All strains contain a gene encoding a secreted, pore-forming toxin known as VacA. Genetic variation at this locus could be under strong selection as H. pylori adapts to the host immune response, colonizes new human hosts, or inhabits different host environments. Here, we analyze the molecular evolution of VacA. Phylogenetic reconstructions indicate the subdivision of VacA sequences into three main groups with distinct geographic distributions. Divergence of the three groups is principally due to positively selected sequence changes in the p55 domain, a central region required for binding of the toxin to host cells. Divergent amino acids map to surface-exposed sites in the p55 crystal structure. Comparative phylogenetic analyses of vacA sequences and housekeeping gene sequences indicate that vacA does not share the same evolutionary history as the core genome. Further, rooting the VacA tree with outgroup sequences from the close relative Helicobacter acinonychis reveals that the ancestry of VacA is different from the African origin that typifies the core genome. Finally, sequence analyses of the virulence determinant CagA reveal three main groups strikingly similar to the three groups of VacA sequences. Taken together, these results indicate that positive selection has shaped the phylogenetic structure of VacA and CagA, and each of these virulence determinants has evolved separately from the core genome.Helicobacter pylori is a Gram-negative bacterium that persistently colonizes the human stomach. H. pylori induces a gastric mucosal inflammatory response known as superficial gastritis and is a risk factor for the development of peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma (2, 43). H. pylori is present in about half of all humans throughout the world.H. pylori strains from unrelated humans exhibit a high level of genetic diversity (5, 44). The population structure of H. pylori is panmictic, and the rate of recombination in H. pylori is reported to be among the highest in the Eubacteria (17, 44). Multilocus sequence analysis of housekeeping genes has revealed the presence of at least nine different H. pylori populations or subpopulations that are localized to distinct geographic regions (12, 27, 31). Analysis of these sequences suggests that H. pylori has spread throughout the world concurrently with the major events of human dispersal, and thus H. pylori is potentially a useful marker for the geographic migrations of human populations (12).One of the important virulence determinants of H. pylori is a secreted toxin known as VacA. VacA is a pore-forming toxin that causes multiple alterations in human cells, including cell vacuolation, depolarization of membrane potential, alteration of mitochondrial membrane permeability, apoptosis, activation of mitogen-activated protein kinases, inhibition of antigen presentation, and inhibition of T-cell activation and proliferation (8, 10, 15). Secreted by an autotransporter (type Va) secretion mechanism, VacA is translated as a 140-kDa protoxin that undergoes N- and C-terminal cleavage during the secretion process to yield an N-terminal signal sequence, a mature 88-kDa secreted toxin known as p88, a small secreted peptide with no known function (termed secreted alpha peptide, or SAP) (7), and a C-terminal beta-barrel domain (41, 47) (Fig. ​(Fig.1A).1A). Two domains of p88 VacA, p33 and p55, have been identified based on partial proteolysis of p88 into fragments of 33 kDa and 55 kDa, respectively (47) (Fig. ​(Fig.1A).1A). The N-terminal p33 domain (residues 1 to 311) is involved in pore formation while the p55 domain (residues 312 to 821) contains one or more cell-binding domains (14, 48). The isolated p55 domain binds to host cells less avidly than does the full-length p88 protein, and in contrast to p88, the isolated p55 domain is not internalized by cells (18, 48). These observations suggest that sequences in both the p33 and p55 domains mediate VacA interactions with the surface of cells.Open in a separate windowFIG. 1.Analysis of VacA phylogeography. (A) The vacA gene encodes a 140-kDa protoxin, which undergoes cleavage to yield a signal sequence, a secreted 88-kDa toxin, a secreted alpha-peptide (SAP), and a C-terminal β-barrel domain. The mature 88-kDa VacA toxin contains two domains, designated p33 and p55. The midregion sequence that defines type m1 and m2 forms of VacA is located within p55. A 21-amino-acid insertion is present in m2 forms but not m1 forms of VacA. (B) Neighbor-joining phylogenetic tree of 100 amino acid sequences of VacA. Three major groups (designated groups 1 to 3) are evident. The chart shows the number of strains analyzed and characteristics of VacA protein sequences in each group of the tree. Group 1 comprises type m1 sequences mainly from non-Asian strains, group 2 comprises m1 sequences from Asian strains, and group 3 comprises m2 sequences from both Asian and non-Asian strains. See Fig. S1 in the supplemental material for a ladder-type version of this tree.All strains of H. pylori contain a chromosomal vacA gene, but individual strains differ considerably in levels of VacA activity (3, 8). Two studies analyzed vacA sequence encoding a fragment of the p33 domain and did not detect any recognizable phylogenetic structure (star or bush-type pattern), presumably due to the presence of extensive recombination (19, 44). Other studies analyzed different regions of VacA and detected polymorphisms that allow classification of vacA alleles into distinct families (designated s1/s2, i1/i2, and m1/m2) depending on the presence of signature sequences in different regions of VacA (3, 4, 39). Geographic differences have been detected within several of these vacA regions (22, 24, 29, 37, 51, 52, 55). In general, strains containing vacA alleles classified as s1, i1, or m1 have been associated with an increased risk of ulcer disease or gastric cancer compared to strains containing vacA alleles classified as s2, i2, or m2 (3, 13, 39).Another important H. pylori virulence factor is the secreted CagA effector protein. The cagA gene is localized within a 40-kb chromosomal region known as the cag pathogenicity island (PAI) (20). H. pylori strains expressing CagA are associated with a significantly increased risk for development of ulcer disease or gastric cancer compared to strains that lack the cagA gene (6). Upon entry into cells, CagA undergoes phosphorylation by host cell kinases and induces numerous alterations in cellular signaling, leading to the designation of CagA as a “bacterial oncoprotein” (20, 32).H. pylori strains that produce an active VacA protein (type s1 VacA) typically express CagA, and strains that produce inactive VacA proteins (type s2 VacA) typically lack the cagA gene (3). vacA and the cag PAI localize to distant sites on the H. pylori chromosome, and, therefore, the basis for this association has been unclear. Recently, several studies have reported that there are complex relationships between the cellular effects of VacA and CagA, whereby VacA can downregulate CagA''s effects on epithelial cells, or vice versa (1, 35, 46, 56). This functional interaction between VacA and CagA may represent a mechanism that allows H. pylori to minimize damage to gastric epithelial cells or minimize mucosal inflammation, thereby allowing it to persistently colonize the stomach.Although VacA is considered an important H. pylori virulence factor and hundreds of studies have classified H. pylori strains based on a vacA typing scheme, there has been very little effort to investigate the forces that drive vacA diversification, to analyze the evolutionary history of vacA, or to correlate vacA diversity with features of the VacA three-dimensional structure. Several important questions remain in studying the vacA gene: (i) Are the s1, i1, and m1 alleles (which are associated with an increased risk of gastroduodenal disease) more recently derived than the s2, i2, and m2 alleles? (ii) Are the geographic differences in vacA alleles driven by adaptive evolution or genetic drift? (iii) Does the evolutionary history of the vacA gene parallel the evolutionary history of the core genes used for MLST analysis, which are markers for ancient migrations of human populations?In the current study, we present a comprehensive analysis of the molecular evolution of vacA. Our analysis of VacA diversity indicates that VacA sequences are clustered into three main groups with distinct geographic distributions. By analyzing topological differences between vacA and housekeeping gene phylogenetic trees, we demonstrate that the vacA gene does not share the same evolutionary history as the core genome of H. pylori. We report that the evolution of VacA has been shaped by positive selection, and adaptive evolution is restricted to the p55 domain. Most of the sequence divergence corresponds to surface-exposed amino acids in the three-dimensional structure of the p55 domain. Finally, we note that there are similarities between the phylogenetic structure of the VacA and CagA trees, and we discuss the roles that positive selection pressures have played in the evolution of these two virulence determinants.     

Seroprevalence of Helicobacter pylori in South Korea

BACKGROUND: Helicobacter pylori-associated gastrointestinal diseases have been widely recognized. The aims of this study were to investigate the interval change of seropositivity of H. pylori between 1998 and 2005 in Korean adult population and to find the factors related to H. pylori infection. METHODS: Between January and December of 2005, a total of 15,916 health check-up subjects (aged > or = 16 years) from all parts of South Korea responded to the questionnaire, and the prevalence of H. pylori was investigated by measuring anti-H pylori IgG antibodies. The seropositivity in asymptomatic subjects (aged > or = 16 years) was compared with that of 1998, which was surveyed by the Korean H. pylori Study Group. RESULTS: The overall seropositivity rate (aged > or = 16 years) was 56.0%, and 13.9% of seropositive subjects were found to have a history of H. pylori eradication therapy. With the exclusion of subjects who had a history of H. pylori eradication and current gastrointestinal symptoms, the seropositivity rate of H. pylori became 59.6% in 8020 subjects. Seroprevalence of H. pylori was significantly higher in subjects aged 50-59 years, males, low income group, and subjects from provinces. The seroprevalence in 2005 (59.6%) significantly decreased compared with that of 1998 (66.9%), and the decrease was significant in subjects aged < 70 years, Seoul and Gyeonggi province (which is close to Seoul). CONCLUSIONS: The seroprevalence of H. pylori in asymptomatic health check-up adult subjects in 2005 decreased to 59.6% from 66.9% in 1998, probably as a result of the improvement of socioeconomic status and hygiene.     

Determination of Helicobacter pylori vacA allelic types by single-step multiplex PCR

AIMS: To develop and evaluate a novel multiplex PCR assay that enables definition of Helicobacter pylori vacA allelic type in a single reaction. METHODS AND RESULTS: Application of the one-step system to DNA extracts from 22 cultures of known vacA genotype demonstrated that it was highly accurate. Analysis of 15 matched gastric biopsy/culture pairs generated exactly correlating genotype profiles. vacA genotypes were determined from an additional 62/70 gastric biopsies from dyspeptic patients of known H. pylori positive status by the one-step assay, compared with 63/70 by the original two-reaction test. Types s1/m1, s1/m2 and s2/m2 were identified in 51.9%, 31.2% and 16.9% of biopsies, respectively. CONCLUSIONS: The multiplex PCR system developed enables rapid one-step vacA genotyping that is accurate, easy to interpret and more economical than the alternative multiple-reaction tests. Application of this system to gastric biopsies from patients in South-east England demonstrated that s1/m1 was the most common genotype, while s1/m2 and s2/m2 were less prevalent. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple one-step system can be applied direct to antral gastric biopsies without the need for culture, thereby facilitating rapid surveillance of vacA genotype in relation to geographical location and disease status.     

The diversity of vacA and cagA genes of Helicobacter pylori in East Asia

It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P=0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P<0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was s1c/m1b. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome.     

Helicobacter pylori vacA and cagA genotypes in patients from northeastern Brazil with upper gastrointestinal diseases

Helicobacter pylori causes chronic gastric inflammation and significantly increases the risk of duodenal and gastric ulcer disease and distal gastric carcinoma. In this study, we evaluated the Helicobacter pylori vacA and cagA genotypes in patients from a Brazilian region where there is a high prevalence of gastric cancer. Polymerase chain reaction (PCR) was used to investigate vacA mosaicism and cagA status in the gastric mucosa of 134 H. pylori-positive patients, including 76 with gastritis: 28 with peptic ulcer disease and 30 with gastric cancer. The s1m1 variant was the predominant vacA genotype observed, whereas the s1 allele was more frequently observed in patients with more severe diseases associated with H. pylori infection [p = 0.03, odds ratio (OR) = 5.72, 95% confidence interval (CI) = 1.15-38.60]. Furthermore, all of the s1 alleles were s1b. Mixed vacA m1/m2 strains were found more frequently in patients with gastric cancer and a cagA-positive status was significantly associated with gastric cancer (p = 0.016, OR = 10.36, 95% CI = 1.35-217.31). Patients with gastric cancer (21/21, 100%, p = 0.006) or peptic ulcers (20/21, 95%, p = 0.02) were more frequently colonised by more virulent H. pylori strains compared to gastritis patients (41/61, 67.2%). In conclusion, in the northeastern of Brazil, which is one of the regions with the highest prevalence of gastric cancer in the country, infection with the most virulent H. pylori strains, carrying the cagA gene and s1m1 vacA alleles, predominates and is correlated with more severe H. pylori-associated diseases.     

Simple sequence repeats in the Helicobacter pylori genome

We describe an integrated system for the analysis of DNA sequence motifs within complete bacterial genome sequences. This system is based around ACeDB, a genome database with an integrated graphical user interface; we identify and display motifs in the context of genetic, sequence and bibliographic data. Tomb et al . (1997) previously reported the identification of contingency genes in Helicobacter pylori through their association with homopolymeric tracts and dinucleotide repeats. With this as a starting point, we validated the system by a search for this type of repeat and used the contextual information to assess the likelihood that they mediate phase variation in the associated open reading frames (ORFs). We found all of the repeats previously described, and identified 27 putative phase-variable genes (including 17 previously described). These could be divided into three groups: lipopolysaccharide (LPS) biosynthesis, cell-surface-associated proteins and DNA restriction/modification systems. Five of the putative genes did not have obvious homologues in any of the public domain sequence databases. The reading frame of some ORFs was disrupted by the presence of the repeats, including the alpha(1-2) fucosyltransferase gene, necessary for the synthesis of the Lewis Y epitope. An additional benefit of this approach is that the results of each search can be analysed further and compared with those from other genomes. This revealed that H . pylori has an unusually high frequency of homopurine:homopyrimidine repeats suggesting mechanistic biases that favour their presence and instability.     

Prevalence of Helicobacter pylori pathogenicity-associated cagA and vacA genotypes among Pakistani dyspeptic patients

The cytotoxin-associated gene A ( cagA ), and the vacuolating cytotoxin gene A ( vacA ) products are considered the most important pathogenic determinants of Helicobacter pylori , a gram-negative bacterium causing gastrointestinal disorders such as duodenal ulcers, gastritis and mucosa-associated lymphoid tissue disease. A higher prevalence of H. pylori has been reported in various regions in the Pakistani population; however, no data are available about the virulence-associated genetic determinants. The objective of this study was to determine the prevalence of virulence-associated genes, cagA, vacA and particularly vacA allelic variants among dyspeptic patients from Pakistan. Gastric biopsy samples were obtained from 78 adult patients presenting dyspepsia symptoms. DNA was isolated and analyzed for the presence of H. pylori and its genotypes by PCR. Genus-specific PCR involving 16S rRNA gene revealed that 66 of the 78 patients were positive for H. pylori , an overall prevalence of 84.6% for this particular study. The most common vacA genotype was s1b/m2 (54.5%) followed by s1a/m1 (19.7%). cagA was positive in 24.2% of the cases and strongly associated with s1a/m1, vacA . The prevalence of virulent cagA , and vacA allelic form s1a/m1 was lower than that reported from neighboring countries.     

Helicobacter pylori bab Paralog Distribution and Association with cagA,vacA, and homA/B Genotypes in American and South Korean Clinical Isolates

Helicobacter pylori genetic variation is a crucial component of colonization and persistence within the inhospitable niche of the gastric mucosa. As such, numerous H. pylori genes have been shown to vary in terms of presence and genomic location within this pathogen. Among the variable factors, the Bab family of outer membrane proteins (OMPs) has been shown to differ within subsets of strains. To better understand genetic variation among the bab genes and to determine whether this variation differed among isolates obtained from different geographic locations, we characterized the distribution of the Bab family members in 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). Overall, we identified 23 different bab genotypes (19 in AH and 11 in KH), but only 5 occurred in greater than 5 isolates. Regardless of strain origin, a strain in which locus A and locus B were both occupied by a bab gene was the most common (85%); locus C was only occupied in those isolates that carried bab paralog at locus A and B. While the babA/babB/- genotype predominated in the KH (78.8%), no single genotype could account for greater than 40% in the AH collection. In addition to basic genotyping, we also identified associations between bab genotype and well known virulence factors cagA and vacA. Specifically, significant associations between babA at locus A and the cagA EPIYA-ABD motif (P<0.0001) and the vacA s1/i1/m1 allele (P<0.0001) were identified. Log-linear modeling further revealed a three-way association between bab carried at locus A, vacA, and number of OMPs from the HOM family (P<0.002). En masse this study provides a detailed characterization of the bab genotypes from two distinct populations. Our analysis suggests greater variability in the AH, perhaps due to adaptation to a more diverse host population. Furthermore, when considering the presence or absence of both the bab and homA/B paralogs at their given loci and the vacA genotype, an association was observed. Our results highlight the multifactorial nature of H. pylori mediated disease and the importance of considering how the specific combinations of H. pylori virulence genes and their multiple interactions with the host will collectively impact disease progression.     

Variations in the gene for vacuolating toxin, vacA, in Japanese isolates of Helicobacter pylori.

Each of 284 strains of Helicobacter pylori which had been isolated in Japan was shown, by use of the polymerase chain reaction (PCR), to be positive for the vacA genes. The amplified vacA genes generated by PCR were classified into six classes (five for the clinical isolates, and one which corresponded to the standard strains). Endoscopic analysis revealed that cases of gastritis were most likely to be associated with class D, while none were associated with class A. The patterns of products of PCR obtained from the Japanese isolates were compared with theoretical patterns derived from sequences of vacA which had been reported previously. The nucleotide sequences of amplified fragments of vacA from representative strains in each class were determined and compared with those of previously reported vacA genes.     

Reduced intracellular survival of Helicobacter pylori vacA mutants in comparison with their wild-types indicates the role of VacA in pathogenesis

The vacuolating cytotoxin VacA of Helicobacter pylori plays an important but yet unknown role in pathogenesis. We studied the impact of the vacuolating cytotoxin on H. pylori invasion of and survival within AGS cells (human gastric cell line derived from an antral adenocarcinoma). Isogenic vacA and cagA mutants were constructed in a wild-type clinical isolate H. pylori, AF4. An H. pylori VacA-deficient mutant, AF4(vacA::kan), was cultured in significantly lower numbers from AGS cells after 24 h incubation with gentamicin added to the culture medium than were the type I wild-type strain AF4 (P<0.03) and an isogenic cagA mutant (P<0.01). Complementation of the AF4 vacA mutant with broth culture supernatant from wild-type AF4 improved the intracellular survival of the vacA mutant. We conclude that H. pylori's vacuolating cytotoxin improves the intracellular survival of H. pylori within AGS cells, suggesting the role of the vacuolating cytotoxin in H. pylori pathogenesis.     

Non-invasive genotyping of Helicobacter pylori cagA, vacA, and hopQ from asymptomatic children

Background: Helicobacter pylori infection is usually acquired in childhood, but little is known about its natural history in asymptomatic children, primarily due to the paucity of non‐invasive diagnostic methods. H. pylori strains harboring cagA and specific alleles of hopQ and vacA are associated with increased risk for gastric cancer. Many studies of H. pylori virulence markers in children have the bias that symptomatic subjects are selected for endoscopy, and these children may harbor the most virulent strains. Our aim is to genotype cagA, hopQ, and vacA alleles in stool DNA samples of healthy Colombian children residing in an area with high incidence of gastric cancer, to avoid selection bias resulting from endoscopy. Methods: H. pylori status of 86 asymptomatic children was assessed by ¹³C‐urea breath test (UBT) and PCR. H. pylori 16S rRNA, cagA, hopQ, and vacA genes were amplified from stool DNA samples and sequenced. Results: UBT was positive in 69 (80.2%) of 86 children; in stool DNA analysis, 78.3% were positive by 16S rRNA PCR. cagA, vacA, and hopQ were detected in 66.1%, 84.6%, and 72.3% of stool DNA samples from 16S rRNA‐positive children. Of the children’s DNA samples, which revealed vacA and hopQ alleles, 91.7% showed vacA s1 and 73.7% showed type I hopQ. Type I hopQ alleles were associated with cagA positivity and vacA s1 genotypes (p < 0.0001). Conclusions: Using stool DNA samples, virulence markers of H. pylori were successfully genotyped in a high percentage of the asymptomatic infected children, revealing a high prevalence of genotypes associated with virulence. Type I hopQ alleles were associated with the presence of cagA and the vacA s1 genotype.     

Clinical relevance of the cagA,vacA and iceA genotypes of Helicobacter pylori in Brazilian clinical isolates

Infection with Helicobacter pylori strains harboring determinants of pathogenicity may lead to a strong inflammatory response in gastric mucosa. In this work, we examined the frequency of the cagA, vacA and iceA genotypes in H. pylori strains isolated from Brazilian patients and correlated these with the clinical manifestations. H. pylori was isolated from 165 patients [30 with non-ulcer dyspepsia cases (NUD); 93 peptic ulcer disease (PUD): 31 gastric ulcers (GU) and 62 duodenal ulcer disease (DU); 18 with erosive gastritis (EG); and 24 gastroesophageal reflux disease (GERD)]. Allelic variants of cagA, vacA and iceA were identified using the polymerase chain reaction. More than one H. pylori strain was detected in 28 cases (17%), and these were excluded from the statistical analysis. We were unable to confirm an association between iceA status and clinical outcome. There was a strong association between the genotype cagA-positive vacA s1 and PUD. However, logistic regression analysis showed that vacA s1 was the only predictive factor for PUD (OR=4.19; 95% CI 1.95-8.98). The presence of the less virulent strain vacA s2 was related to GERD (OR=8.59; 95% CI 2.85-25.91). Our results support the hypothesis that virulent strains may protect against the development of GERD.     

cagA gene and vacA alleles in Spanish Helicobacter pylori clinical isolates from patients of different ages

The prevalence of the cagA gene and vacA alleles in 124 Spanish Helicobacter pylori clinical isolates from patients of different ages ranging from 3 to 78 years was studied (21 patients < or = 10 years, 30 patients 11-20 years, 17 patients 21-40 years, 31 patients 41-60 years and 25 patients 61-80 years). The cagA gene and vacA s1 or vacA s2 alleles were identified by PCR from the strain. 66.9% of the isolates were cagA+ and 33.1% cagA-. vacA s1 was detected in 48.4% of the isolates and vacA s2 in 51.6%. 44.4% of patients were cagA+/vacA s1, 22.5% were cagA+/vacA s2, 4% were cagA-/vacA s1 and 29% were cagA-/vacA s2. The percentage of cagA+ isolates and the vacA s1 alleles in the different groups were as follows: 23.8% and 28.6% in 0-10 years, 40% and 30% in 11-20 years, 88.2% and 70.6% in 21-40 years, 90.3% and 70.9% in 41-60 years and 92% and 44% in the 61-78 years group. 93% (54/58) of isolates found in ulcer patients and 90.9% (10/11) of isolates from gastritis patients older than 20 years were cagA+. In patients younger than 20 years ulcer disease was rare with 60% of isolates being cagA+ (3/5) compared with 31.6% cagA+ isolates (12/38) in patients suffering from gastritis in the younger group. The prevalence of the cagA gene and vacA s1 allele increased with age, being more frequent in older patients than in younger.     

A protocol for isolating putative Helicobacter pylori from fecal specimens and genotyping using vacA alleles

Background. This study outlines steps for isolating and culturing Helicobacter pylori from freshly voided fecal specimens and genotyping isolates for vacA alleles. Materials and methods. A family with four H. pylori‐infected members participated in this pilot study. Criterion for participation was a positive test for H. pylori by the urea breath test. Fecal specimens from children were taken from a freshly soiled diaper, placed in cold buffer, and prepared for culture in less than 2 hours. Culturing of H. pylori utilized selective culture media and isolates were screened for negative Gram stain, positive catalase and oxidase tests, and positive H. pylori 16S ribosomal RNA polymerase chain reaction (PCR). Strain types were determined by vacA genotyping. Results. The isolation procedure is relatively simple, although 5–7 days are required for H. pylori culturing. Isolation and purification of DNA eliminated PCR inhibitors and resulted in reliable analyses. All four family members were infected with the same H. pylori strain with a genotype of vacA s1a/m2. Conclusion. This research lays the foundation for developing a routine and direct noninvasive method to detect the presence of H. pylori in fecal specimens. It is especially convenient for diagnosing children and infants, as samples can be obtained from soiled diapers. Culturing H. pylori from fecal samples in certain cases is important for antibiotic resistant studies prior to treating infected patients and for strain genotyping in epidemiological studies to determine transmission.     

Diversity of Helicobacter pylori cagA and vacA genes in Costa Rica: its relationship with atrophic gastritis and gastric cancer

BACKGROUND: Associations between Helicobacter pylori gene diversity and gastric cancer have not been reported on in Costa Rica, despite its being one of the countries with the highest gastric cancer incidence and mortality rates in the world. The aim of this study was to determine the prevalence of H. pylori cagA and vacA genes and investigate whether it could be correlated with atrophic gastritis (AG) and gastric cancer (GC) in Costa Rica. MATERIALS AND METHODS: Genomic DNAs from isolates of 104 patients classified into two groups: non-atrophic gastritis group (n = 68) and atrophic gastritis group (n = 36), were subjected to PCR-based genotyping of cagA and vacA genes and their correlation with clinical outcome was investigated. Total DNA extractions from gastric tissues of 25 H. pylori-infected gastric cancer patients were utilized for comparative purposes. RESULTS: The presence of cagA (75.3%), vacA s1b (75.3%), and vacA m1 (74.2%) was detected, and colonization by strains with different vacA genotypes in the same stomach was found in 9.7% of the patients. Age- and sex-adjusted vacA s1b and vacA m1 were associated with GC while only vacA m1 was significantly associated with AG. A tendency for association between cagA and vacA s1b, and AG was reported. CONCLUSIONS: The prevalence status of the cagA and vacA (s1/m1) genes in Costa Rica seems to fall between that found in European/North American and East Asian countries, and both cagA and vacA seem to have clinical relevance in this country.