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1.  Diversity of Helicobacter pylori genotypes in Iranian patients with different gastroduodenal disorders 
AIM: To investigate the diversity of Helicobacter pylori (H. pylori) genotypes and correlations with disease outcomes in an Iranian population with different gastroduodenal disorders.
METHODS: Isolates of H. pylori from patients with different gastroduodenal disorders were analyzed after culture and identification by phenotypic and genotypic methods. Genomic DNA was extracted with the QIAamp DNA mini kit (Qiagen, Germany). After DNA extraction, genotyping was done for cagA, vacA (s and m regions), iceA (iceA1, iceA2) and babA with specific primers for each allele using polymerase chain reaction (PCR). All patients’ pathologic and clinical data and their relation with known genotypes were analyzed by using SPSS version 19.0 software. χ2 test and Fisher’s exact test were used to assess relationships between categorical variables. The level of statistical significance was set at P < 0.05.
RESULTS: A total of 71 isolates from 177 patients with different gastroduodenal disorders were obtained. Based on analysis of the cagA gene (positive or negative), vacA s-region (s1 or s2), vacA m-region (m1 or m2), iceA allelic type (iceA1 and iceA2) and babA gene (positive or negative), twenty different genotypic combinations were recognized. The prevalence of cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, iceA1+iceA2 and babA were 62%, 78.9%, 19.7%, 21.1%, 78.9%, 15.5%, 22.5%, 40.8% and 95.8%, respectively. Interestingly, evaluation of PCR results for cagA in 6 patients showed simultaneous existence of cagA variants according to their size diversities that proposed mixed infection in these patients. The most prevalent genotype in cagA-positive isolates was cagA+/vacAs1m2/iceA1+A2/babA+ and in cagA-negative isolates was cagA-/vacAs1m2/iceA-/babA+. There were no relationships between the studied genes and histopathological findings (H. pylori density, neutrophil activity, lymphoid aggregation in lamina propria and glandular atrophy). The strains which carry cagA, vacAs1/m1, iceA2 and babA genes showed significant associations with severe active chronic gastritis (P = 0.011, 0.025, 0.020 and 0.031, respectively). The vacAs1 genotype had significant correlation with the presence of the cagA gene (P = 0.013). Also, babA genotype showed associations with cagA (P = 0.024). In the combined genotypes, only cagA+/vacAs1m1/iceA2/babA+ genotype showed correlation with severe active chronic gastritis (P = 0.025).
CONCLUSION: This genotyping panel can be a useful tool for detection of virulent H. pylori isolates and can provide valuable guidance for prediction of the clinical outcomes.
doi:10.3748/wjg.v19.i34.5685
PMCID: PMC3769906  PMID: 24039362
Helicobacter pylori; cagA; vacA; iceA; babA
2.  Genotyping CagA, VacA subtype, IceA1, and BabA of Helicobacter pylori isolates from Korean patients, and their association with gastroduodenal diseases. 
Journal of Korean Medical Science  2001;16(5):579-584.
The genetic status of cagA, vacA subtype, iceA1, and babA, and the relationship to gastroduodenal diseases were assessed in Helicobacter pylori isolates in Korea. Seventy-six strains of H. pylori were isolated from the antrum and the corpus of 41 adult patients (22 with peptic ulcer and 19 with gastritis). The cagA, iceA1, and babA genes were assessed by polymerase chain reaction and the vacA subtypes were determined by reverse hybridization-line probe assay. The positive rates of 349-bp cagA, 208-bp cagA, iceA1, and babA genes were 97.4%, 96.1%, 84.2%, and 36.1%, respectively. The vacA s1a, s1b, s1c, and s2 variants were detected in 11.8%, 3.9%, 80.4%, and 1.3%, respectively. m1 (78.9%) is more prevalent than m2 (5.3%). The most common vacA genotype was s1c/m1 (61.9%), and 14 isolates (18.4%) contained mixed vacA genotypes from a single biopsy specimen. Twenty-one (60%) of 35 patients were infected with more than two strains of different cagA, iceA1, babA, and vacA genotypes. None of cagA, iceA1, babA, and vacA s1/m1 were associated with peptic ulcer. In conclusion, most H. pylori isolates in Korea carry cagA, iceA1, and vacA s1c/m1 genes, and reside with multiple strains. These genes do not correlate with the peptic ulcer in the Korean patients.
PMCID: PMC3057590  PMID: 11641526
3.  Analysis of iceA genotypes in South African Helicobacter pylori strains and relationship to clinically significant disease 
Gut  2001;49(5):629-635.
BACKGROUND—South African Helicobacter pylori isolates are characterised by the universal presence of cagA but have differences in vacuolating cytotoxin gene (vacA) alleles which correlate with clinically significant disease. However, the candidate virulence marker gene iceA has not been investigated.
AIM—To characterise the genetic organisation and heterogeneity of iceA genotypes in different South African clinical isolates.
PATIENTS AND METHODS—We studied H pylori strains isolated from 86 dyspeptic patients (30 with peptic ulcer disease (PUD), 19 with distal gastric adenocarcinoma (GC), and 37 with non-erosive gastritis) for the presence of iceA1 or iceA2 genes, and for differences in the genetic organisation of iceA2 by polymerase chain reaction, Southern hybridisation analysis, and sequencing.
RESULTS—Genetic analysis of iceA1 demonstrated significant homology (92-95%) with the USA type strain 26695 and probably functions as a transcriptional regulator, while a novel variant (iceA2D') of iceA2 and marked differences in predicted protein secondary structure of the iceA2 protein were defined. iceA1 was detected in 68% and iceA2 in 80% of all clinical isolates. Although approximately 40% of patients had both strains, a higher prevalence (p< 0.01) of GC patients were infected with iceA1 isolates which were invariably vacA s1/iceA1 (p< 0.005 v gastritis). Isolates from PUD patients were distinguished by the structurally altered iceA2D variant (53%; p<0.03 v gastritis) while the iceA2C variant distinguished isolates from patients with gastritis alone (67%; p< 0.005 v PUD).
CONCLUSION—In this study, an association between iceA1 and GC was noted while differences in variants of iceA2 differentiated between PUD and gastritis alone. Combination analyses of iceA genotypes and vacA alleles supported these associations.


Keywords: adenocarcinoma; gastritis; Helicobacter pylori; iceA; peptic ulceration; protein prediction; sequencing; South Africa
doi:10.1136/gut.49.5.629
PMCID: PMC1728508  PMID: 11600464
4.  Helicobacter pylori in Vegetables and Salads: Genotyping and Antimicrobial Resistance Properties 
BioMed Research International  2014;2014:757941.
From a clinical and epidemiological perspective, it is important to know which genotypes and antibiotic resistance patterns are present in H. pylori strains isolated from salads and vegetables. Therefore, the present investigation was carried out to find this purpose. Three hundred eighty washed and unwashed vegetable samples and fifty commercial and traditional salad samples were collected from Isfahan, Iran. Samples were cultured and those found positive for H. pylori were analyzed using PCR. Antimicrobial susceptibility testing was performed using disk diffusion method. Seven out of 50 (14%) salad and 52 out of 380 (13.68%) vegetable samples harbored H. pylori. In addition, leek, lettuce, and cabbage were the most commonly contaminated samples (30%). The most prevalent virulence genes were oipA (86.44%) and cagA (57.625). VacA s1a (37.28%) and iceA1 (47.45%) were the most prevalent genotypes. Forty different genotypic combinations were recognized. S1a/cagA+/iceA1/oipA+ (33.89%), s1a/cagA+/iceA2/oipA (30.50%), and m1a/cagA+/iceA1/oipA+ (28.81%) were the most prevalent combined genotypes. Bacterial strains had the highest levels of resistance against metronidazole (77.96%), amoxicillin (67.79%), and ampicillin (61.01%). High similarity in the genotyping pattern of H. pylori among vegetable and salad samples and human specimens suggests that vegetable and salads may be the sources of the bacteria.
doi:10.1155/2014/757941
PMCID: PMC4145543  PMID: 25184146
5.  Relationship between Helicobacter pylori iceA, cagA, and vacA Status and Clinical Outcome: Studies in Four Different Countries 
Journal of Clinical Microbiology  1999;37(7):2274-2279.
There is continuing interest in identifying Helicobacter pylori virulence factors that might predict the risk for symptomatic clinical outcomes. It has been proposed that iceA and cagA genes are such markers and can identify patients with peptic ulcers. We compared H. pylori isolates from four countries, looking at the cagA and vacA genotypes, iceA alleles, and presentation of the infection. We used PCR to examine iceA, vacA, and cagA status of 424 H. pylori isolates obtained from patients with different clinical presentations (peptic ulcer, gastric cancer, and atrophic gastritis). The H. pylori isolates examined included 107 strains from Bogota, Colombia, 70 from Houston, Tex., 135 from Seoul, Korea, and 112 from Kyoto, Japan. The predominant genotype differed among countries: the cagA-positive iceA1 vacA s1c-m1 genotype was predominant in Japan and Korea, the cagA-positive iceA2 vacA s1b-m1 genotype was predominant in the United States, and the cagA-positive iceA2 vacA s1a-m1 genotype was predominant in Colombia. There was no association between the iceA, vacA, or cagA status and clinical outcome in patients in the countries studied. iceA status shows considerable geographic differences, and neither iceA nor combinations of iceA, vacA, and cagA were helpful in predicting the clinical presentation of an H. pylori infection.
PMCID: PMC85136  PMID: 10364597
6.  Association of peptic ulcer with increased expression of Lewis antigens but not cagA, iceA, and vacA in Helicobacter pylori isolates in an Asian population 
Gut  2000;47(1):18-22.
BACKGROUND—Studies in Western populations suggest that cagA, iceA, and vacA gene status in Helicobacter pylori isolates is associated with increased virulence and peptic ulcer disease.
AIM—To investigate the relationship between peptic ulcer and expression of Lewis (Le) antigens as well as cagA, iceA, and vacA in H pylori isolates in Singapore.
METHODS—Expression of Le antigens in H pylori isolates obtained from patients with dyspepsia was measured by enzyme linked immunosorbent assay. The cagA, iceA, and vacA status was determined by polymerase chain reaction.
RESULTS—Of 108 H pylori isolates, 103 (95.4%) expressed Lex and/or Ley, while Lea and Leb were expressed in 23 (21.3%) and 47 (43.5%) isolates, respectively. Expression of two or more Le antigens (Lex, Ley, Lea, or Leb) was significantly higher in H pylori isolated from ulcer patients than in non-ulcer patients (89.6% v 73.2%, p=0.035). There were no significant differences in the prevalence of cagA or iceA1 in H pylori isolates from peptic ulcer and non-ulcer patients (86.6% v 90.2% for cagA; 70.1% v 68.3% for iceA1), and no association of peptic ulcer with any specific vacA genotype.
CONCLUSIONS—The present study indicates that peptic ulcer disease is associated with increased expression of Lewis antigens but not cagA, iceA, or vacA genotype in H pylori isolates in our population. This suggests that cagA, iceA, and vacA are not universal virulence markers, and that host-pathogen interactions are important in determining clinical outcome.


Keywords: Lewis blood group antigens; cagA; iceA; vacA; Helicobacter pylori; peptic ulcer
doi:10.1136/gut.47.1.18
PMCID: PMC1727977  PMID: 10861258
7.  H pylori iceA alleles are disease-specific virulence factors 
AIM: To characterize and compare genotype profiles of H pylori strains isolated from patients with chronic gastritis and duodenal ulcer in western part of Turkey.
METHODS: A total of 46 patients [30 chronic gastritis (CG) and 16 duodenal ulcer (DU)] who had undergone endoscopy because of dyspeptic complaints were studied. The antral biopsy specimens were evaluated for the presence of H pylori by rapid urease test and culture, and the genotype profiles were determined by real-time PCR.
RESULTS: The cagA gene was observed in 43 (93.5%) isolates. The vacA s1m2 genotype was the predominant subtype, found in 63.3% and 68.7% of isolates in patients with CG and DU, respectively. Twenty (66.6%) isolates from patients with CG were iceA2 positive while the iceA1 was predominant in those with DU (68.8%). In terms of the association of the iceA alleles to other genes, both alleles were significantly associated with the cagA vacA s1m2 genotype.
CONCLUSION: The prevalent circulating genotypes in CG and DU were cagA vacA s1m2 iceA2 and cagA vacA s1m2 iceA1 genotype, respectively. It was found that cagA vacA s1m2 genotype seems to be common virulence factors in both CG and DU while iceA alleles show specificity for gastroduodenal pathologies in this study.
doi:10.3748/wjg.v13.i18.2581
PMCID: PMC4146818  PMID: 17552005
H pylori; Virulence genes; Culture; Real-time PCR
8.  Prevalence of Helicobacter pylori virulence genotypes among children in Eastern Turkey 
AIM: To identify the virulence genotypes of Helicobacter pylori (H. pylori) if present in children in Eastern Turkey and if those genotypes are mostly associated with severe clinical presentations.
METHODS: A total of 49 H. pylori positive Turkish children (42 with antral nodularity and 7 with peptic ulcer) who underwent upper gastrointestinal endoscopy with abdominal symptoms during the period from March 2011 to September 2012 were enrolled in this study. Antral nodularity was diagnosed endoscopically by two of the authors. We determined for the presence of cagA, vacA, cagE, iceA and babA2 genotypes of H. pylori isolates in DNA obtained directly from frozen gastric biopsy samples by polymerase chain reaction test using specific primers.
RESULTS: Of the 49 H. pylori isolates studied, 61.2%, 91.8%, 22.4%, 28.6%, 57.1% and 40.8% were positive for the cagA, vacA s1, cagE, iceA1, iceA2 and babA2 genes, respectively. We showed that the most common vacA subtype was s1a (79.6%). However, the s2 gene was found less frequently with an isolation rate of 8.2% of the H. pylori isolates. The genotypes iceA2 and vacA s1m2 were the most frequently found types in children with antral nodularity. In addition, the genotypes iceA1, babA2 and vacA s1m1 were found in similar ratios in all the H. pylori isolates obtained from children with peptic ulcer. The genotypes vacA s2m1 and s1c were not observed in any of isolates studied.
CONCLUSION: This study showed that vacA s1m2, cagA and iceA2 were the most common genotypes, and no association between antral nodularity and genotypes was observed.
doi:10.3748/wjg.v19.i39.6585
PMCID: PMC3801372  PMID: 24151385
Helicobacter pylori; Children; Genotype; Polymerase chain reaction
9.  Characterization of Helicobacter pylori cagA and vacA Genotypes among Alaskans and Their Correlation with Clinical Disease▿ 
Journal of Clinical Microbiology  2011;49(9):3114-3121.
Helicobacter pylori infection is common in Alaska. The development of severe H. pylori disease is partially determined by the virulence of the infecting strain. Here we present vacA and cagA genotype data for H. pylori strains isolated from Alaskans and their correlation with clinical disease. We enrolled patients scheduled for esophagogastroduodenoscopy and positive for H. pylori infection. Gastric biopsy specimens from the stomach antrum and fundus were cultured. We performed PCR analysis of the H. pylori vacA gene and for the presence of the cagA gene and cagA empty site. We genotyped 515 H. pylori samples from 220 Native and 66 non-Native Alaskans. We detected the cagA gene in 242/286 (85%) persons; of 222 strains that could be subtyped, 95% (212) were non-Asian cagA and 3% (6) were East Asian cagA. After removing mixed infections (n = 17), 83% of H. pylori strains had either the vacA s1m1 (120/269) or s2m2 (103/269) genotype. Sixty-six percent (68/103) of H. pylori strains with the vacA s2m2 genotype also contained the cagA gene. Infection with an H. pylori strain having the cagA gene or vacA s1m1 genotype (compared with s1m2 and s2m2) was associated with a decreased risk of esophagitis (P = 0.003 and 0.0003, respectively). Infection with an H. pylori strain having the vacA s1m1 genotype (compared with s1m2 and s2m2) was associated with an increased risk of peptic ulcer disease (PUD) (P = 0.003). The majority of H. pylori strains in this study carried the non-Asian cagA gene and either the vacA s1m1 or s2m2 genotype. A majority of H. pylori strains with the vacA s2m2 genotype also contained the cagA gene. There was an association of H. pylori genotype with esophagitis and PUD.
doi:10.1128/JCM.00469-11
PMCID: PMC3165571  PMID: 21752979
10.  Regulation of Gastric Carcinogenesis by Helicobacter pylori Virulence Factors 
Cancer research  2008;68(2):379-387.
Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, and strains that possess the cag secretion system, which translocates the bacterial effector CagA into host cells, augment cancer risk. H. pylori strains that express the vacuolating cytotoxin or the outer membrane protein OipA are similarly associated with severe pathologic outcomes. We previously reported that an in vivo adapted H. pylori strain, 7.13, induces gastric adenocarcinoma in rodent models of gastritis. In the current study, we used carcinogenic strain 7.13 as a prototype to define the role of virulence constituents in H. pylori–mediated carcinogenesis. Mongolian gerbils were infected with wild-type strain 7.13 or cagA−, vacA−, or oipA− mutants for 12 to 52 weeks. All infected gerbils developed gastritis; however, inflammation was significantly attenuated in animals infected with the cagA− but not the vacA− or oipA− strains. Gastric dysplasia and cancer developed in >50% of gerbils infected with either the wild-type or vacA− strain but in none of the animals infected with the cagA− strain. Inactivation of oipA decreased β-catenin nuclear localization in vitro and reduced the incidence of cancer in gerbils. OipA expression was detected significantly more frequently among H. pylori strains isolated from human subjects with gastric cancer precursor lesions versus persons with gastritis alone. These results indicate that loss of CagA prevents the development of cancer in this model. Inactivation of oipA attenuates β-catenin nuclear translocation and also decreases the incidence of carcinoma. In addition to defining factors that mediate H. pylori–induced cancer, these results provide insight into mechanisms that may regulate the development of other malignancies arising within the context of inflammatory states.
doi:10.1158/0008-5472.CAN-07-0824
PMCID: PMC3114460  PMID: 18199531
11.  Comparative genomics of Helicobacter pylori isolates recovered from ulcer disease patients in England 
BMC Microbiology  2005;5:32.
Background
Genomic diversity of H. pylori from many different human populations is largely unknown. We compared genomes of 65 H. pylori strains from Nottingham, England. Molecular analysis was carried out to identify rearrangements within and outside the cag-pathogenicity-island (cag PAI) and DNA sequence divergence in candidate genes. Phylogenetic analysis was carried out based on various high-resolution genotyping techniques.
Results
Analyses of virulence genes (cagT, cagE, cagA, vacA, iceA, oipA and babB) revealed that H. pylori strains from England are genetically distinct from strains obtained from other countries. The toxigenic vacA s1m1 genotype was found to be less common and the plasticity region cluster was found to be disrupted in all the isolates. English isolates showed a predominance of iceA1 alleles and a functional proinflammatory oipA gene. The English H. pylori gene pool revealed several Asian/oriental features. This included the predominance of cagA – glr (cagA right junction) motif types III and II (up to 42%), presence of vacA m1c alleles and phylogenetic affinity towards East Asian / Amerindian gene pools based on fluorescent amplified fragment length polymorphism (FAFLP) analysis and glmM sequence analysis.
Conclusion
Overall, our results demonstrated genetic affinities of H. pylori in England with both European and the Asian gene pools and some distinctive genetic features of virulence genes that may have evolved in this important European population.
doi:10.1186/1471-2180-5-32
PMCID: PMC1180443  PMID: 15916705
12.  Determination of Helicobacter pylori Virulence Genes in Gastric Biopsies by PCR 
ISRN Gastroenterology  2013;2013:606258.
Aim. The aim of this study was to identify the presence of H. pylori in biopsy specimens from symptomatic patients by PCR. In addition, the rate of cagA, vacA, iceA1, and iceA2 virulence genes was determined. Materials and Methods. One hundred antral gastric biopsy specimens were collected during endoscopy from patients suffering from gastroduodenal symptoms. The samples were collected by the gastroenterologists in their own clinics in Ramallah, Palestine. DNA was extracted from the biopsies and subsequently used for PCR identification of H. pylori and the virulence genes using specific primers. Results. The rate of positive H. pylori in the collected biopsies was 44%. The rates of the virulence genes in this sample: cagA, vacA, iceA1, and iceA2 were 65.9%, 40.9%, 63.6%, and 84.1%, respectively. Conclusion. The iceA2 gene was the most frequent in this study. Much research is necessary to determine the presence of an association of this gene with gastric pathology. Variation in the rates of the iceA gene in different countries is a strong indication of its geographical distribution. This study would provide important information regarding the prevalence of virulence genes (vacA, cagA, iceA1, and iceA2) in H. pylori strains in the sample tested in this country.
doi:10.1155/2013/606258
PMCID: PMC3649278  PMID: 23691338
13.  Distribution of Helicobacter pylori cagA, cagE, oipA and vacA in different major ethnic groups in Tehran, Iran 
Background and Aim
There are geographical variations in Helicobacter pylori virulence genes; cagA, cagE, vacA and oipA. The present study compared the distribution of these genotypes in major ethnic groups residing in Tehran, Iran and their association with clinical outcomes.
Methods
A total of 124 H. pylori-positive patients living in Tehran were enrolled in this study. The ethnic distribution was 74 Persians, 33 Turks and 17 other ethnics including Kurds, Lurs, Afghanis and Arabs. The presence of the cagA, cagE and oipA genes and vacA alleles (signal [s] and middle [m] region) were determined by polymerase chain reaction (PCR) from H. pylori DNA.
Results
The cagA-positive status was predominant in all three ethnic groups (e.g. 65% in Persians and 73% in Turks). In contrast, the cagE-positive status was less than half in Persians (47%) and Turks (30%), whereas it was 77% in other ethnicities (P = 0.008). The predominant vacA genotypes were s1 and m1 in all three ethnic groups (e.g. 68% in Persians and 70% in Turks were s1). There was no significant association between cagA and cagE status or vacA genotypes and clinical outcomes. The oipA-positive strains were more common in non-ulcer dyspepsia (NUD) (63%) than in peptic ulcer patients (15%) (P = 0.001) in Persians, but the association was not observed in other ethnic groups.
Conclusion
There are some differences in the H. pylori genotypes among the ethnic groups in Iran. However, none of these markers seemed to be clinically helpful in predicting the clinical presentation of a H. pylori infection in Iran.
doi:10.1111/j.1440-1746.2009.05876.x
PMCID: PMC3128249  PMID: 19702906
clinical outcome; ethnic; Helicobacter pylori genotype; Iran; virulence factor
14.  Prevalence of genotypes in Helicobacter pyloriisolates from patients in eastern Turkey and the association of these genotypes with clinical outcome 
Brazilian Journal of Microbiology  2012;43(4):1332-1339.
There is not much information available regarding the prevalence of the genotypes of Helicobacter pylori isolates in Turkey, particularly in eastern Turkey. The aims of this study were to detect the prevalence of different genotypes of H. pylori in Turkish patients with gastrointestinal complaints and to determine the relationship of these genotypes with clinical outcome and sex. One hundred forty H. pylori isolates were examined for the presence of its genotypes by the PCR. We found that the prevalence of vacAs1,vacAs2, cagA, cagE, iceA1, iceA2 and babA2 genes were 88.6%, 11.4%, 71.4%, 35.7%, 41.4%, 58.6%, and 62.1%, respectively. The most predominant vacA subtype was s1a (81.4%). The most vacA allelic combination detected were vacAs1m1 (65.2%) and s1m2 (53.9%) in patients with peptic ulcer and gastritis, respectively. The only vacAs1 isolate was significantly associated with gastritis and peptic ulcer (p<0.05). The vacAs1a, ml, slml and babA2 genes were significantly associated with peptic ulcer (p<0.05), whereas m2 gene was significantly associated with only gastritis (p<0.05). The difference between sex and genotypes was statistically significant among the cagA,vacAs1, iceA2 and babA2 genes. This study reported for the first time the prevalence of H. pylori genotypes in patients with gastrointestinal complaints in eastern Turkey. Further studies are needed to understand epidemiological importance of the genotypes of H. pylori isolates in this region and the association between the virulence genes and clinical outcome in different regions.
doi:10.1590/S1517-838220120004000014
PMCID: PMC3769024  PMID: 24031961
Helicobacter pylori; prevalence; patient; genotypes; PCR
15.  Analysis of antimicrobial susceptibility and virulence factors in Helicobacter pylori clinical isolates 
BMC Gastroenterology  2003;3:20.
Background
In this study, we evaluated the prevalence of primary resistance of Brazilian H. pylori isolates to metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone. In addition, the vacA, iceA, cagA and cagE genotypes of strains isolated from Brazilian patients were determined and associated with clinical data in an effort to correlate these four virulence markers and antibiotic resistance.
Methods
H. pylori was cultured in 155 H. pylori-positive patients and MICs for metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone were determined by the agar dilution method. Genomic DNA was extracted, and allelic variants of vacA, iceA, cagA and cagE were identified by the polymerase chain reaction.
Results
There was a strong association between the vacA s1/cagA -positive genotype and peptic ulcer disease (OR = 5.42, 95% CI 2.6–11.3, p = 0.0006). Additionally, infection by more virulent strains may protect against GERD, since logistic regression showed a negative association between the more virulent strain, vacA s1/cagA-positive genotype and GERD (OR = 0.26, 95% CI 0.08–0.8, p = 0.03). Resistance to metronidazole was detected in 75 patients (55%), to amoxicillin in 54 individuals (38%), to clarithromycin in 23 patients (16%), to tetracycline in 13 patients (9%), and to furazolidone in 19 individuals (13%). No significant correlation between pathogenicity and resistance or susceptibility was detected when MIC values for each antibiotic were compared with different vacA, iceA, cagA and cagE genotypes.
Conclusion
The analysis of virulence genes revealed a specific association between H. pylori strains and clinical outcome, furthermore, no significant association was detected among pathogenicity and resistance or susceptibility.
doi:10.1186/1471-230X-3-20
PMCID: PMC194586  PMID: 12911839
16.  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.
doi:10.1128/CDLI.9.1.167-175.2002
PMCID: PMC119904  PMID: 11777849
17.  Clinical and histological associations of cagA and vacA genotypes in Helicobacter pylori gastritis. 
Journal of Clinical Pathology  1998;51(1):55-61.
AIMS: To determine the relation among the cytotoxin associated gene (cagA) and vacuolating cytotoxin gene (vacA) status of Helicobacter pylori isolates, the associated clinical diseases, and the severity and pattern of chronic gastritis. METHODS: Helicobacter pylori was cultured from gastric biopsies obtained from dyspeptic patients. DNA was extracted from the isolates and the cagA and vacA status determined by the polymerase chain reaction (PCR). The prevalence of the different cagA and vacA genotypes in three clinical groups, duodenal ulcer, gastric ulcer, and non-ulcer dyspepsia was compared. The histological features in sections from two antral and two corpus biopsies were graded by one blinded observer. The grades were compared with age and sex matched groups with different cagA and vacA genotypes, and with duodenal ulcers, or non-ulcer dyspepsia. RESULTS: Isolates from 161 patients were included. One hundred and nine (68%) harboured a cagA+ strain and 143 (89%) harboured a vacA s1 strain. The prevalence of cagA+ strains in duodenal ulcer patients (94%) was highly significantly greater than in those with non-ulcer dyspepsia (56%). However, of the patients infected with a cagA+ strain, almost equal numbers had non-ulcer dyspepsia or peptic ulceration. Chronic inflammation, polymorph activity, surface epithelial degeneration, atrophy, and intestinal metaplasia were all significantly more severe in the cagA+ than in the cagA- group, whereas only corpus epithelial degeneration was significantly more severe in the vacA s1 group compared with the vacA s2 group. Patients infected with cagA+ strains were almost four times more likely to have antral intestinal metaplasia than cagA- patients. An antral predominant gastritis was present in duodenal ulcer patients compared with matched non-ulcer dyspepsia patients, but this was not attributable to cagA or vacA status. CONCLUSIONS: Helicobacter pylori strains showing cagA positively and the vacA s1 genotype are associated with more severe gastritis but these virulence factors do not appear to determine the overall pattern. The pattern is closely linked to clinical disease. Therefore, it is likely that the nature of the disease complicating chronic infection is determined by host and environmental factors, while bacterial factors determine the magnitude of the risk of developing such disease.
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PMCID: PMC500433  PMID: 9577374
18.  Evaluation of the Association of Nine Helicobacter pylori Virulence Factors with Strains Involved in Low-Grade Gastric Mucosa-Associated Lymphoid Tissue Lymphoma  
Infection and Immunity  2004;72(2):880-888.
Helicobacter pylori has been associated with the development of two malignant diseases: gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Although the cag pathogenicity island, especially the cagA gene, has been linked with adenocarcinoma, few data concerning H. pylori pathogenic factors involved in low-grade gastric MALT lymphoma are available. The goal of this study was to analyze the prevalence of and correlation between genes coding for seven H. pylori virulence factors (cagA, cagE, vacA, iceA, babA, hopQ, and oipA) and two novel adhesins (sabA and hopZ) by comparing a collection of 43 H. pylori strains isolated from patients with low-grade gastric MALT lymphoma to 39 strains isolated from age-matched patients with gastritis only. Our results show that taken individually, none of the nine genes tested can be considered associated with MALT strains and allow us to conclude that MALT pathogenesis is not linked with more proinflammatory H. pylori strains. We demonstrated that in patients infected with strains harboring the iceA1 allele, sabA functional status, and hopZ “off” status, the odds of developing a MALT lymphoma were 10 times higher. However, the low prevalence of such strains (10 of 43 MALT strains) renders this triple association a low-sensitivity marker for MALT strains. Our data confirmed that H. pylori virulence factors are correlated with one another. If the involvement of H. pylori in MALT lymphoma is well established, the pathomechanism by which gastric lymphoma occurs remains to be identified.
doi:10.1128/IAI.72.2.880-888.2004
PMCID: PMC321584  PMID: 14742532
19.  The Efficacy of Laboratory Diagnosis of Helicobacter pylori Infections in Gastric Biopsy Specimens Is Related to Bacterial Density and vacA, cagA, and iceA Genotypes 
A total of 500 consecutive patients undergoing upper endoscopy were biopsied and tested for H. pylori infection by the Campylobacter-like organism (CLO) test, culture, histology, and PCR. Serum samples were tested by two different serological assays. Patients were considered H. pylori positive if at least two of the four biopsy specimen-based methods yielded positive results. PCR had the highest diagnostic sensitivity (99.4%), followed by histology (92.2%), culture (89.5%), and the CLO test (89.0%). The specificities of all methods were higher than 98%. Of the organisms from the 181 PCR-positive patients, the vacA (s and m regions), cagA, and iceA genotypes were determined by reverse hybridization (line probe assay) or an allele-specific PCR. Organisms that were detected by PCR but that remained undetected by the CLO test were significantly more often vacA s1 (P = 0.006), m1 (P = 0.028), and cagA positive (P = 0.029) than vacA s2, m2, and cagA negative, respectively. Organisms that were detected by PCR but that remained undetected by culture or histology more often contained iceA1 (P = 0.034 and P = 0.029, respectively) than iceA2. Higher H. pylori density was associated with vacA s2 (P = 0.024), vacA m2 (P = 0.050), and cagA-negative (P = 0.035) genotypes. Also, the diagnostic results of the CLO test (P = 0.001) and culture (P = 0.031) but not those of the PCR (P = 0.130) were significantly associated with the H. pylori density. The rate of detection by the four biopsy specimen-based tests was lower for patients who used proton pump inhibitors, but this was independent of the H. pylori genotypes. These observations may be explained by different bacterial densities, as established by the distinct genotypes of H. pylori, and confirm that the biologies of strains with such genotypes are considerably different.
PMCID: PMC86006  PMID: 10618055
20.  Investigation of the association between clinical outcome and the cag pathogenicity-island and other virulence genes of Helicobacter pylori isolates from patients with dyspepsia in Eastern Turkey 
Brazilian Journal of Microbiology  2014;44(4):1267-1274.
The aims of our work were to determine the presence of the cag pathogenicity-island (cag PAI) and other virulence genes of Helicobacter pylori recovered from patients with gastritis and peptic ulcer, and to investigate the correlation of these virulence genes with clinical outcome. The presence of the cagA, the promoter regions of cagA, cagE, cagT, and the left end of cag-PAI (LEC), cag right junction (cagRJ), the plasticity region open reading frames (ORFs), vacA and oipA genes among 69 H. pylori isolates were determined by polymerase chain reaction. Intact cag PAI was detected in only one (1.4%) isolate. The cagA gene was identified in 52.1% and 76.2% of isolates from patients with dyspepsia (gastritis and peptic ulcer), respectively. The plasticity region ORFs i.e. JHP912 and JHP931 were predominantly detected in isolates from peptic ulcer. Less than 25% of the isolates carried other ORFs. Types I, II and III were the most commonly found among the isolates. None of the isolates possessed type Ib, 1c, IIIb, IV and V motifs. The most commonly vacA genotypes were s1am1a and s1m2 in isolates with peptic ulcer and gastritis, respectively. The results confirmed that the prevalence of oipA (Hp0638) gene was 75% and 85.7% in patients with gastritis and peptic ulcer, respectively. Furthermore, vacA s1am1a positivity was significantly related to peptic ulcer (p < 0.05).
PMCID: PMC3958197  PMID: 24688521
Helicobacter pylori; gastritis; peptic ulcer; cag pathogenicity-island; polymerase chain reaction
21.  Association between Helicobacter pylori genotypes and severity of chronic gastritis, peptic ulcer disease and gastric mucosal interleukin-8 levels: Evidence from a study in the Middle East 
Gut Pathogens  2014;6(1):41.
Background
The varied clinical presentations of Helicobacter pylori (H. pylori) infection are most likely due to differences in the virulence of individual strains, which determines its ability to induce production of interleukin-8 (IL-8) in the gastric mucosa. The aim of this study was to examine association between cagA, vacA-s1 and vacA-s2 genotypes of H. pylori and severity of chronic gastritis and presence of peptic ulcer disease (PUD), and to correlate these with IL-8 levels in the gastric mucosa.
Methods
Gastric mucosal biopsies were obtained from patients during esophagogastroduodenoscopy. The severity of chronic gastritis was documented using the updated Sydney system. H. pylori cagA and vacA genotypes were detected by PCR. The IL-8 levels in the gastric mucosa were measured by ELISA.
Results
H. pylori cagA and/or vacA genotypes were detected in 99 patients (mean age 38.4±12.9; 72 males), of whom 52.5% were positive for cagA, 44.4% for vacA-s1 and 39.4% for vacA-s2; and 70.7% patients had PUD. The severity of inflammation in gastric mucosa was increased with vacA-s1 (p=0.017) and decreased with vacA-s2 (p=0.025), while cagA had no association. The degree of neutrophil activity was not associated with either cagA or vacA-s1, while vacA-s2 was significantly associated with decreased neutrophil activity (p=0.027). PUD was significantly increased in patients with cagA (p=0.002) and vacA-s1 (p=0.031), and decreased in those with vacA-s2 (p=0.011). The level of IL-8 was significantly increased in patients with cagA (p=0.011) and vacA-s1 (p=0.024), and lower with vacA-s2 (p=0.004). Higher levels of IL-8 were also found in patients with a more severe chronic inflammation (p=0.001), neutrophil activity (p=0.007) and those with PUD (p=0.001).
Conclusions
Presence of vacA-s1 genotype of H. pylori is associated with more severe chronic inflammation and higher levels of IL-8 in the gastric mucosa, as well as higher frequency of PUD. Patients with vacA-s2 have less severe gastritis, lower levels of IL-8, and lower rates of PUD. The presence of cagA genotype is not associated with the severity of gastritis or IL-8 induction in the gastric mucosa. The association of cagA with PUD may be a reflection of its presence with vacA-s1 genotype.
doi:10.1186/s13099-014-0041-1
PMCID: PMC4181383  PMID: 25279005
Helicobacter pylori; cagA; vacA-s1; vacA-s2; Gastritis; Interleukin-8; Peptic ulcer disease
22.  Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases 
Journal of Clinical Microbiology  1998;36(4):944-948.
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.
PMCID: PMC104665  PMID: 9542913
23.  Prevalence of Helicobacter pylori vacA, cagA and iceA genotypes and correlation with clinical outcome 
The aim of this study was to assess the genetic status of cagA, vacA subtype and iceA genotypes of Helicobacter pylori and the relationship with upper gastrointestinal diseases in Northeast China. Gastric biopsies were obtained from 378 patients with upper gastrointestinal diseases and 197 samples were used. The cagA, vacA alleles and iceA genotypes were determined by polymerase chain reaction. CagA was present in 176 (89.3%) of 197 patients. Of the 197 cases, 186 (94.4%) had vacA signal sequence s1c allele, 6 (3%) had s1a and 5 (2.5%) had s1b. The vacA s2 genotype was not detected in our study. VacA middle region sequences, m1 and m2, were found in 20 (10.2%) and 150 (76.1%), respectively. The allelic variant iceA1 (70.1%) was more prevalent than iceA2 (23.4%). The vacA allele s1am2 had a significant relationship with the presence of gastric cancer (p<0.05) and the iceA1 genotype was also associated with gastric cancer (p<0.05). These may be useful risk factors for upper gastrointestinal diseases.
doi:10.3892/etm.2012.704
PMCID: PMC3494117  PMID: 23226771
Helicobacter pylori; prevalence; genotype; Northeast China
24.  Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan 
Gut  1998;42(3):338-343.
Background—VacA and CagA proteins have been reported to be major virulence factors of Helicobacter pylori. However, antibodies against these proteins are frequently found in the sera of Japanese patients regardless of their gastroduodenal status. 
Aim—To evaluate the expression of VacA and CagA proteins by H pylori strains isolated in Japan. 
Methods—By using specific antibodies raised against recombinant VacA and CagA proteins, the expression of VacA and CagA was evaluated in 68 H pylori strains isolated from Japanese patients; a vacuolating assay and genotyping of the vacA gene were also used in the evaluation. The results were analysed in relation to the gastroduodenal diseases of the hosts. 
Results—VacA and CagA proteins were expressed in 59/68 (87%) and in 61/68 (90%) isolates respectively. The vacuolating assay was positive in 57/68 (84%) isolates, indicating that most immunologically VacA positive strains produced active cytotoxin. The prevalence of infection with strains expressing CagA and positive for vacuolating activity (Type I) was very high, 54/68 (79%), irrespective of the gastroduodenal status of the host. 
Conclusion—Most H pylori isolates in Japan are positive for vacuolating cytotoxin and CagA, and thus these virulence factors cannot be used as markers to discern the risk of developing serious gastroduodenal pathologies in the hosts. However, the high prevalence of infection with strains positive for vacuolating cytotoxin and CagA may contribute to the characteristics of H pylori infection in Japan. 


Keywords: VacA; Helicobacter pylori; CagA; ulcer; non-ulcer dyspepsia
PMCID: PMC1727030  PMID: 9577338
25.  Role of Helicobacter pylori cagA EPIYA motif and vacA genotypes for the development of gastrointestinal diseases in Southeast Asian countries: a meta-analysis 
BMC Infectious Diseases  2012;12:223.
Background
Infection with cagA-positive, cagA EPIYA motif ABD type, and vacA s1, m1, and i1 genotype strains of Helicobacter pylori is associated with an exacerbated inflammatory response and increased risk of gastroduodenal diseases. However, it is unclear whether the prevalence and virulence factor genotypes found in Southeast Asia are similar to those in Western countries. Here, we examined the cagA status and prevalence of cagA EPIYA motifs and vacA genotypes among H. pylori strains found in Southeast Asia and examined their association with gastroduodenal disease.
Methods
To determine the cagA status, cagA EPIYA motifs, and vacA genotypes of H. pylori, we conducted meta-analyses of 13 previous reports for 1,281 H. pylori strains detected from several Southeast Asian countries.
Results
The respective frequencies of cagA-positive and vacA s1, m1, and i1 genotypes among examined subjects were 93% (1,056/1,133), 98% (1,010/1,033), 58% (581/1,009), and 96% (248/259), respectively. Stratification showed significant variation in the frequencies of cagA status and vacA genotypes among countries and the individual races residing within each respective country. The frequency of the vacA m-region genotype in patients infected with East Asian-type strains differed significantly between the northern and southern areas of Vietnam (p < 0.001). Infection with vacA m1 type or cagA-positive strains was associated with an increased risk of peptic ulcer disease (odds ratio: 1.46, 95%CI: 1.01-2.12, p = 0.046 and 2.83, 1.50-5.34, p = 0.001, respectively) in the examined Southeast Asian populations.
Conclusions
Both Western- and East Asian-type strains of H. pylori are found in Southeast Asia and are predominantly cagA-positive and vacA s1 type. In Southeast Asia, patients infected with vacA m1 type or cagA-positive strains have an increased risk of peptic ulcer disease. Thus, testing for this genotype and the presence of cagA may have clinical usefulness.
doi:10.1186/1471-2334-12-223
PMCID: PMC3519793  PMID: 22994150
Helicobacter pylori; cagA; vacA; EPIYA; Gastric cancer; Peptic ulcer disease; Southeast Asian

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