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A novel method for genotyping the Helicobacter pylori vacA intermediate region directly in gastric biopsy specimens.

ABSTRACT: 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.

SUBMITTER: Ferreira RM

PROVIDER: S-EPMC3502994 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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A novel method for genotyping the Helicobacter pylori vacA intermediate region directly in gastric biopsy specimens.

Ferreira Rui M RM Machado Jose C JC Letley Darren D Atherton John C JC Pardo Maria L ML Gonzalez Carlos A CA Carneiro Fatima F Figueiredo Ceu C

Journal of clinical microbiology 20121003 12

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 ...[more]

PMID: 23035185

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Project description:A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD450S) of > or = 3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD450S equal to or greater than the cutoff (mean OD450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD450, 2.910 +/- 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD450, 0.108 +/- 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD450S were > or = 3.0. The colorimetric hybridization assay also detected two other H. pylori-positive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.

Dataset Information

A novel method for genotyping the Helicobacter pylori vacA intermediate region directly in gastric biopsy specimens.

Publications

A novel method for genotyping the Helicobacter pylori vacA intermediate region directly in gastric biopsy specimens.

Similar Datasets

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