Validation of a Novel Immunoline Assay for Patient Stratification according to Virulence of the Infecting Helicobacter pylori Strain and Eradication Status.
ABSTRACT: Helicobacter pylori infection shows a worldwide prevalence of around 50%. However, only a minority of infected individuals develop clinical symptoms or diseases. The presence of H. pylori virulence factors, such as CagA and VacA, has been associated with disease development, but assessment of virulence factor presence requires gastric biopsies. Here, we evaluate the H. pylori recomLine test for risk stratification of infected patients by comparing the test score and immune recognition of type I or type II strains defined by the virulence factors CagA, VacA, GroEL, UreA, HcpC, and gGT with patient's disease status according to histology. Moreover, the immune responses of eradicated individuals from two different populations were analysed. Their immune response frequencies and intensities against all antigens except CagA declined below the detection limit. CagA was particularly long lasting in both independent populations. An isolated CagA band often represents past eradication with a likelihood of 88.7%. In addition, a high recomLine score was significantly associated with high-grade gastritis, atrophy, intestinal metaplasia, and gastric cancer. Thus, the recomLine is a sensitive and specific noninvasive test for detecting serum responses against H. pylori in actively infected and eradicated individuals. Moreover, it allows stratifying patients according to their disease state.
Project description:Helicobacter pylori colonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome. H. pylori virulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to important H. pylori virulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed in Escherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) were H. pylori negative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), the recomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, the recomLine assay provides a valuable tool for the diagnosis of H. pylori infection.
Project description:Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA(+)/VacA(+)H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.
Project description:Incidence and mortality rates for gastric cancer, the fifth most commonly diagnosed and third most deadly cancer worldwide, are highest in East Asia. We sought to identify gastric cancer risk biomarkers among eight prospective studies from China, Japan and Korea.This pooled nested case-control study included 1608 incident non-cardia gastric cancer cases and 1958 matched controls. Pre-diagnostic antibody levels to 15 Helicobacter pylori proteins were assessed using multiplex serology. Conditional logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs).Sero-positivity to 10 H. pylori antigens (Omp, CagA, VacA, HcpC, HP 0305, GroEL, NapA, HyuA, Cad, HpaA) was associated with a 1.29- to 3.26-fold increase in odds of gastric cancer. Omp and HP 0305 consistently remained associated with gastric cancer risk after mutually adjusting for all other markers. Sero-positivity to both Omp and HP 0305 was associated with an over 4-fold increase in gastric cancer incidence (OR, 4.09; 95% CI 3.26-5.13). When limited to only those who are CagA+ H. pylori+, Omp/HP 0305 sero-positivity remained strongly associated with an over 3-fold increase in the odds of gastric cancer (OR, 3.34; 95% CI 2.27-4.91). The results were highly consistent among the cohorts.We have confirmed new H. pylori biomarkers that are strongly associated with gastric cancer risk, even among those infected with the known H. pylori virulence factor CagA. These results may help to design cost-efficient prevention strategies to reduce gastric cancer incidence in East Asia.
Project description:Helicobacter pylori (H. pylori) is the causative agent of gastric cancer, making it the only bacterium to be recognized as a Class I carcinogen by the World Health Organization. The virulence factor cytotoxin associated gene A (CagA) is a known oncoprotein that contributes to the development of gastric cancer. The other major virulence factor vacuolating cytotoxin A (VacA), disrupts endolysosomal vesicular trafficking and impairs the autophagy pathway. Studies indicate that there is a functional interplay between these virulence factors by unknown mechanisms. We show that in the absence of VacA, both host-cell autophagy and the proteasome degrade CagA during infection with H. pylori. In the presence of VacA, CagA accumulates in gastric epithelial cells. However, VacA does not affect proteasome function during infection with H. pylori suggesting that VacA-disrupted autophagy is the predominant means by which CagA accumulates. Our studies support a model where in the presence of VacA, CagA accumulates in dysfunctional autophagosomes providing a possible explanation for the functional interplay of VacA and CagA.
Project description: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.H. pylori status of 86 asymptomatic children was assessed by (13) C-urea breath test (UBT) and PCR. H. pylori 16S rRNA, cagA, hopQ, and vacA genes were amplified from stool DNA samples and sequenced.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).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.
Project description:Bangladesh has a population with a low gastric cancer risk but high prevalence of Helicobacter pylori infection. Several studies have examined virulence genes in H. pylori from Bangladesh. We analyzed cagA and vacA subtypes and their association with severe histology phenotypes, and analyzed population types among Bangladeshi strains. We included patients who underwent endoscopy in Dhaka. Sequences of virulence genes and seven housekeeping genes were obtained by next generation sequencing and confirmed by Sanger sequencing. We isolated 56 H. pylori strains from 133 patients, of which 73.2% carried cagA, and all were considered Western-type. Patients infected with cagA-positive strains had more severe histological scores than patients infected with cagA-negative strains. Among vacA s1 and m1 genotypes, the s1a (97.8%, 43/44) and m1c (28/30, 93.3%) genotypes were predominant. All strains containing s1 and m1 (30/56, 53.6%) also had i1, d1, and c1. In contrast, all strains containing the less-virulent genotypes s2 and m2 (12/56, 21.4%) also possessed i2, d2, and c2. Multivariate analysis indicated that subjects infected with vacA m1-genotype strains only had a significantly higher risk of antrum atrophy than patients infected with m2-genotype strains. Of the two main H. pylori populations in this study, hpAsia2 strains were associated with higher activity and inflammation in the antrum compared to hpEurope strains; however, only vacA s1m1i1d1c1 strains, independent of population type, were significantly associated with inflammation in the antrum, unlike the s2m2i2d2c2 genotype. In conclusion, Bangladeshi strains were divided into two main populations of different genotypes. The low incidence of gastric cancer in Bangladesh might be attributable to the high proportion of less-virulent genotypes, which may be a better predictor of gastric cancer risk than the ancestral origin of the H. pylori strains. Finally, the vacA m region may be a better virulence marker than other regions.
Project description:Objective:The clinical outcomes of gastric diseases such as chronic gastritis, peptic ulcer, and gastric cancer have been attributed to the interplay of virulence factors of Helicobacter pylori (H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of cagA, vacA, iceA2, babA2, and oipA genes and their association with clinical outcomes. Methods:Chronic gastritis, atrophic gastritis, and intestinal metaplasia specimens were obtained from patients who underwent endoscopy and surgical resection between January 2017 and December 2018; specimens from gastric cancer patients treated between January 2014 and December 2018 were also added. H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of cagA, vacA, iceA2, babA2, and oipA genes and their association with clinical outcomes. H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of. Results:H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of vacA, babA2, and oipA genes and their association with clinical outcomes. vacA, babA2, and oipA genes and their association with clinical outcomes. P=0.033, OR?=?2.64; 95% CI?=?1.44-4.82, P=0.033, OR?=?2.64; 95% CI?=?1.44-4.82, P=0.033, OR?=?2.64; 95% CI?=?1.44-4.82, H. pylori vacA +/babA2, and oipA genes and their association with clinical outcomes. P=0.033, OR?=?2.64; 95% CI?=?1.44-4.82. Conclusion:In this present study, we reported on the virulence genes of H. pylori infection to reveal their association with increased risk of chronic gastritis, precancerous gastric lesions, and gastric cancer. Precancerous gastric lesions with H. pylori vacA+/babA2+/oipA+ genotype increased the risk of gastric cancer.H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of H. pylori vacA +/babA2, and oipA genes and their association with clinical outcomes.
Project description:The present report describes a novel method for genotyping the virulence-associated vacA intermediate (i) region of Helicobacter pylori in archive material. vacA i-region genotypes as determined by the novel method were completely concordant with those of sequence analysis and with those of functional vacuolation activity. The method was further validated directly in gastric biopsy specimens of 386 H. pylori-positive cases, and effective characterization of the vacA i region was obtained in 191 of 192 (99.5%) frozen and in 186 of 194 (95.9%) formalin-fixed paraffin-embedded gastric biopsy specimens, respectively. The genotyping method was next used to address the relationship between the vacA genotypes and the cagA status. The vacA i1 genotype was associated with vacA s1 (where s indicates signal region), vacA m1 (where m indicates middle region), and cagA-positive genotypes (P < 0.0001), while the vacA i2 genotype was closely related with vacA s2, vacA m2, and cagA-negative genotypes (P < 0.0001). The relationship between H. pylori vacA i-region genotypes and gastric disease development was subsequently evaluated in the Portuguese population. Patients infected with vacA i1 strains showed an increased risk for gastric atrophy and for gastric carcinoma, with odds ratios of 8.0 (95% confidence interval [CI], 2.3 to 27) and of 22 (95% CI, 7.9 to 63), respectively. Taken together, the results show that this novel H. pylori vacA i-region genotyping method can be applied directly to archive material, providing a fast evaluation of strain virulence determinants without the need of culture. The results further emphasize that the characterization of the vacA i region may be useful to identify patients at higher risk of gastric carcinoma development.
Project description:Helicobacter pylori (H. pylori) is a well-recognized gastroduodenal pathogen and class I carcinogen. Dual oxidase-2 (DUOX2), a member of NADPH oxidase family, has several critical physiological functions, including thyroid hormone biosynthesis and host mucosal defense.To investigate the effect of H. pylori infection on DUOX2 gene expression in human stomach.The biopsies were obtained from patients who underwent endoscopic diagnosis. The patient serum was assayed for two virulence factors of H. pylori, CagA IgG and VacA. The inflammation in gastric mucosa was analyzed with histology. Real-time quantitative PCR was used to detect the expression of three members of NADPH oxidase, NOX1, NOX2, and DUOX2, as well as lactoperoxidase (LPO) in the gastric mucosa. NOX2, DUOX2, and myeloperoxidase (MPO) protein levels were quantified by Western blots or immunohistochemistry.The H. pylori-infected gastric mucosa had more severe inflammation than uninfected samples. However, the expression of DUOX2 mRNA and protein was lower in gastric mucosa of patients with H. pylori infection compared to the uninfected. Among the H. pylori-infected patients, those having CagA IgG or VacA in the serum had lower DUOX2 expression levels than those infected with H. pylori without either virulence factor. The NOX2 and MPO levels were higher in those patients infected with H. pylori irrespective of the virulence factors than those uninfected patients. NOX1 and LPO mRNA were undetectable in the gastric mucosa.CagA+ or VacA+ H. pylori in the stomach of patients may suppress DUOX2 expression to promote its own survival. Increased NOX2 could not eliminate H. pylori infection.
Project description:Helicobacter pylori is a pathogenic bacterium that causes various gastrointestinal diseases. The most common gastric malignancies associated with H. pylori are gastric cancer and lymphoma of mucosa associated lymphoid tissue (MALT). Helicobacter pylori virulence genes, namely cagA and vacA, are known to be associated with malignancy development. Conventionally, cagA and vacA were classified by looking at partial sequences of the genes. However, such genotyping has hardly proven useful predicting different risks for gastric cancer or MALT lymphoma. In search of new loci that distinguish these diseases, we investigated the full sequences of cagA and vacA.We compared cagA and vacA sequences of 18 and 12 H. pylori strains obtained, respectively, from patients with gastric cancer and MALT lymphoma in Oita, Japan. Conventional genotyping of cagA and vacA showed no significant difference between the two diseases. We further investigated the full protein sequences of CagA and VacA to identify loci where allele frequency was significantly different between the diseases. We found four such loci on CagA, and three such loci on VacA. We also inspected the corresponding loci on the genes of 22 gastritis strains that potentially lead to gastric cancer or MALT lymphoma in the long run. Significant differences were observed at one CagA locus between gastritis and MALT lymphoma strains, and at one VacA locus between gastritis and gastric cancer strains.We found novel candidate loci in H. pylori virulence genes in association with two different types of gastric malignancies that could not be differentiated by conventional genotyping. Biological connotations of the amino acid polymorphisms merit further study.