Helicobacter pylori and cagA and vacA gene status in children from Brazil with chronic gastritis

Abstract. Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1–16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

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

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

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.     

Evaluation of a Helicobacter pylori Stool Antigen Test for the Diagnosis and Follow-Up of Infections in Children

 The aim of this study was to evaluate the performance of a newly developed enzyme immunoassay kit (HpSA) for detecting Helicobacter pylori antigens in the stool of children. This study was comprised of 58 children referred to various endoscopy units for evaluation of gastrointestinal symptoms and upper gastroduodenal endoscopy and 11 children for post-therapy follow-up. In the first group, 23 children were diagnosed as positive for Helicobacter pylori using bacteriological and/or histological methods. Stool antigens were detected in 20 of these positive patients, for a sensitivity of 86.9% and a negative predictive value of 91.9%. Since only one false-positive reaction was observed with the HpSA kit, the specificity was 97.1% and the positive predictive value 95.2%. Results obtained for post-therapy follow-up were also promising. The HpSA assays were negative for the eight children whose infections were eradicated after therapy, and a positive result was obtained for two of three patients who had a persistent infection.     

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

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

Frequency of vacA Genotypes and Cytotoxin Activity in Helicobacter pylori Associated with Low-Grade Gastric Mucosa-Associated Lymphoid Tissue Lymphoma

The vacA genotypes s1,m1 and s1,m2 were detected in 44 and 30% of Helicobacter pylori isolates, respectively, from patients with gastric mucosa-associated lymphoid tissue lymphoma, compared to 26 and 56% of isolates, respectively, from individuals with gastritis. The vacA s1 genotype was significantly associated with, but not predictive of, the presence of vacuolating cytotoxin activity.     

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.     

A new histological procedure for re-evaluation of the serological test forHelicobacter pylori

To re-evaluate the accuracy of the serological test forHelicobacter pylori, fixation of biopsy specimens with Carnoy's solution (preserving the mucous layer in tissue preparations) followed by immunohistochemical staining (a new histological procedure) was used as the reference histological method instead of 10% formalin fixation followed by hematoxylin-eosin staining (the conventional histological procedure). Biopsy specimens (antrum and body) from 114 patients with gastritis (including non-ulcer dyspepsia) or gastric and/or duodenal ulcers were obtained by endoscopy and used for both bacteriological culture and histological examination. Serum samples were taken from all patients at the time of endoscopy. The serum levels of specific IgG and IgA antibodies forHelicobacter pylori were measured by commercial enzyme immunoassay kits. The reliability of the IgG and IgA measurements was evaluated by analyzing receiver operating characteristic curves obtained using the two histological procedures. With the conventional histological procedure as the reference, the sensitivity and specificity levels of the serological test were 87.2% and 82.1%, respectively. With the new histological procedure as reference, sensitivity and specificity were 94% and 96.7%, respectively. The insufficient accuracy reported for the serological test could be due to false-positive or false-negative results obtained when the conventional histological procedure is used as the reference. The new histological procedure used here revealed that the serological test forHelicobacter pylori is more reliable than previously thought.     

Expanding Allelic Diversity of Helicobacter pylori vacA

The diversity of the gene encoding the vacuolating cytotoxin (vacA) of Helicobacter pylori was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions of vacA, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from H. pylori isolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different vacA s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (cagA). This PCR-LiPA method permits rapid analysis of the vacA and cagA status of H. pylori strains for clinical and epidemiological studies and will facilitate identification of any further variations.     

Cytotoxin production by Helicobacter pylori from patients with upper gastrointestinal tract diseases.

A cytotoxin produced by some Helicobacter pylori strains has recently been identified. The cytotoxin induces intracellular vacuolization of cultured cells. The aim of the present study was to examine the frequency of occurrence of cytotoxin-producing strains of H. pylori from subjects with upper gastrointestinal disease including nonulcer dyspepsia, gastric and duodenal ulcer disease, gastroesophageal reflux disease, and gastric cancer. Broth culture filtrates of clinical isolates of H. pylori recovered from 175 patients were used to inoculate Vero and HeLa cell monolayers for the detection of vacuolating cytotoxin activity. The results obtained demonstrated that the highest percentage of strains producing cytotoxin were found in subjects with peptic ulcer disease (gastric ulcer, 65%; duodenal ulcer, 66%; P < 0.01 compared with nonulcer dyspepsia, 38%). Of the 11 patients with gastroesophageal reflux disease, 4 of 5 patients in this group who had esophageal ulcers, were found to be infected with strains that produced cytotoxin. Three of the four patients with carcinoma of the stomach were also found to be infected with cytotoxic strains of H. pylori. With increasing severity of mucosal damage in subjects with a normal upper gastrointestinal tract, macroscopic gastritis, duodenitis, and peptic ulceration, there were corresponding increase in the proportion of strains producing cytotoxin; these increases were 32, 46, 50, and 66%, respectively. H. pylori strains from subjects with ulcer disease commonly produced vacuolating cytotoxin, suggesting that it may be a virulence factor in the pathogenesis of peptic ulcer disease.     

cag Pathogenicity island-dependent upregulation of matrix metalloproteinase-7 in infected patients with Helicobacter pylori

Helicobacter pylori (H. pylori) infection has been involved in the pathogenesis of most important gastroduodenal diseases. Matrix metalloproteinases (MMPs) are a large family of zincendopeptidases which play important roles in degradation of extracellular matrix (ECM) and various inflammatory diseases. Therefore, we examined MMP-7 mRNA levels in the gastric mucosa of patients with H. pylori infection and evaluated the effects of virulence factors, such as vacA (vacuolating cytotoxin A) and cagA (cytotoxin-associated gene), in H. pylori-infected patients upon the MMP-7 mRNA mucosal levels. We also determined the correlation between mucosal MMP-7 mRNA levels and the types of disease. Total RNA was extracted from gastric biopsies of 50 H. pylori-infected patients and 50 uninfected individuals. Mucosal MMP-7 mRNA expression level in H. pylori-infected and non-infected gastric biopsies was determined by real-time polymerase chain reaction (PCR). The presences of cagA and vacA virulence factors was evaluated using PCR. MMP-7 expression was significantly higher in biopsies of patients infected with H .pylori compared to uninfected individuals. In addition, mucosal MMP-7 mRNA expression in H. pylori-infected patients significantly associated with the cagA status and the types of disease. Our results suggest that MMP-7 might be involved in the pathogenesis of H. pylori. Peptic ulcer was associated with cag pathogenicity island-dependent MMP-7 upregulation.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> Virulence Genotypes in Portuguese Children and Adults with Gastroduodenal Pathology

The aim of this study was to evaluate the prevalence of virulence genotypes, namely cagA, vacA and babA2, of Helicobacter pylori strains isolated from Portuguese adults and children presenting gastroduodenal pathology. One hundred thirty-six strains were studied, 82 isolated from adult patients (50 with nonulcerative gastritis and 32 with active peptic ulcer) and 58 isolated from children (54 with nonulcerative gastritis and 4 with duodenal ulcer). Genotyping of cagA, vacA and babA2 was assessed by polymerase chain reaction. Overall, Helicobacter pylori strains carrying more virulent genotypes were much more prevalent in adults than in children, particularly the type I (vacAs1- and cagA-positive) and the triple-positive (vacAs1-, cagA- and babA2-positive) strains (P<0.001). A subpopulation of adults and children with nonulcerative gastritis was also studied, and differences in the prevalence of virulent genotypes were observed, either for individual genotypes (P=0.017 for cagA, P=0.010 for vacAs1) or in combinations, i.e. the type I genotype (P=0.005) and the triple-positive strains (P=0.031). There was no difference between the two populations in the distribution of babA2 and m1/m2 genotypes. Considering the cohort effect in the epidemiology of Helicobacter pylori infection, these results suggest that different strains might circulate during different periods of time, or that, after infection in childhood, individual strains will undergo changes during the course of infection. Electronic Publication     

Vacuolating cytotoxic activity of 40 Helicobacter pylori strains isolated from Turkish patients

In this study we examined the in vitro vacuolating cytotoxic activity of Helicobacter pylori, which is a gram-negative microaerophilic curved bacterium and a causative agent of gastritis, peptic ulcer and gastric ulcer. A vacuolating cytotoxin assay was performed to assess the vacuolating activity of 40 strains (20 gastritis, 11 gastric ulcer, and 9 duodenal ulcer), which were obtained from patients undergoing upper gastrointestinal endoscopy. The Vero cell line was used in the cytotoxic assay. Of the 40 isolates, 24 (12 gastritis, 6 gastric ulcer, 6 duodenal ulcer) were cytotoxic for the Vero cell line at 1:4 and 1:8 dilutions. Thus, vacuolating cytotoxin of H. pylori affects the Vero cell line, but it seems there is no correlation between the positivity of the strains and the risk of any particular H. pylori disease.     

Possible evidence of invasiveness ofHelicobacter (Campylobacter)pylori

Gastric/duodenal biopsy material from 52 patients was examined immunohistochemically forHelicobacter (Campylobacter) pylori. Specimens from 34 of the patients harbouredHelicobacter pylori along the mucosal surface and 13 of these featured, in addition, immunopositive material within the lamina propria. The remaining 18 biopsies were non-reactive. This observation suggests thatHelicobacter pylori can penetrate the epithelium and its basement membrane, resulting in the production of specific systemic antibodies.     

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⁺.     

Transport conditions and number of biopsies necessary for culture ofHelicobacter pylori

Stuart's transport medium with a charcoal impregnated swab was tested for transport of biopsies from the gastric antrum for culture ofHelicobacter pylori. Biopsies were cultured under microaerophilic conditions either within 2 h or after a delay of 24 h at 4 °C. In 65 patients referred for gastroscopy two biopsies were taken.Helicobacter pylori was found in 39 patients. The rate of survival ofHelicobacter pylori was found to be as high in Stuart's transport medium after 24 h at 4 °C as in the paired biopsy which was cultured immediately after transportation in normal saline. In five (13 %) of 39 patients positive forHelicobacter pylori, the organism was cultured from only one of the biopsies. It is therefore recommended that two biopsies be taken for culture.     

Detection ofHelicobacter pylori in gastric biopsy tissue by polymerase chain reaction

To evaluate the sensitivity of a polymerase chain reaction (PCR) assay using nested primers in detectingHelicobacter pylori, gastric tissue biopsy specimens were collected on endoscopy from 17 patients with a duodenal ulcer. DNA was extracted by phenol/chloroform treatment or boiling in water, and then subjected to a nested PCR using two primer pairs from the urease gene ofHelicobacter pylori. Fourteen of the 17 patients were positive forHelicobacter pylori using DNA samples extracted by either method. The PCR results correlated well with the results of an enzyme immunoassay to detect IgG antibody. However, there were two culture negative patients. The three PCR negative patients were both culture negative and serologically negative. DNA from 9 of the 14 patients was randomly selected and subjected to semiquantification by serial dilutions, and then PCR. The results showed that phenol/chloroform extraction yielded 10–1000 times more DNA than the boiling method. It is concluded that the PCR assay is a rapid and sensitive method for detectingHelicobacter pylori, and that phenol/chloroform extraction is superior to simple boiling in obtaining DNA samples for PCR.     

Neutralisation of cytotoxic vacuolating activity by serum antibodies of Helicobacter pylori-infected patients

The study involved 196 H. pylori strains and 196 serum samples taken from the same patients. H. pylori strains were investigated for the production of vacuolating cytotoxin. Antibodies to the vacuolating cytotoxin produced by H. pylori were detected in the sera samples by neutralisation assay (on Intestine 407 cells) and ELISA. Of the 196 H. pylori strains tested, 80 (40.8%) were found to express vacuolating cytotoxic activity. The titres of cytotoxic nonconcentrated broth culture filtrates ranged from 1:2 to 1:128. The vacuolating assay was positive in 37.1% strains isolated from children, and in 50% strains isolated from adults. Cytotoxin-positive H. pylori strains were found more frequently in duodenal ulcer (71%) than in chronic gastritis (35.2%) patients, and this difference was statistically significant p < 0.05. Neutralising antibodies to vacuolating cytotoxin were present in 51% and 49% of the serum samples tested by neutralisation and ELISA, respectively. Duodenal ulcer patients had antibodies to vacuolating cytotoxin more frequently (p < 0.05) than chronic gastritis patients. Antibodies to cytotoxin were detected in the serum samples from patients infected by cytotoxic (100%) and noncytotoxic (18%) H. pylori strains.     

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.