The aim of this study was to evaluate whether PCR-based restriction fragment length polymorphism (RFLP) analysis was effective in differentiating between reinfection and recrudescence of H. pylori strains. Following a 1-2 week regimen of omeprazole 20 mg, amoxicillin 1.0 g, and clarithromycin 500 mg twice daily, twenty patients with duodenal ulcer were enrolled in the study. Ten patients (group 1, control) were not successfully treated, and another 10 patients (group 2) exhibited recurrence of infection 6-24 months following the therapy. Follow-up diagnosis was performed by Giemsa stain and CLO test. RFLP profiles of antral and midbody biopsy specimens were compared before and after therapy. PCR products using the ureC gene were digested with restriction enzymes Hha I, Mbo I, and Hind III, and the fragments generated were analyzed by agarose gel electrophoresis. Hha I, Mbo I, and Hind III digestion produced 13, 7, and 2 distinguishable digestion patterns, respectively. There was no difference in RFLP profiles seen before and after the therapy in 17 duodenal ulcer patients, while different RFLP profiles were discovered in 3 patients. Following treatment, one (group 2) patient differed in Mbo I, and two (one each from both groups) patients differed in Hha I and Mbo I RFLP patterns. Eight of group 2 patients showed recrudescence of previous infection and two patients had reinfection by another strain. This study supports the hypothesis that PCR-based RFLP analysis can be effective for differentiating reinfection and recrudescence of H. pylori strains following triple therapy.
Adult ; Female ; Helicobacter Infections/drug therapy ; Helicobacter Infections/diagnosis* ; Helicobacter pylori/isolation & purification* ; Helicobacter pylori/genetics ; Human ; Male ; Polymerase Chain Reaction* ; Polymorphism, Restriction Fragment Length* ; Recurrence

OBJECTIVES: Considering the geographic differences in the prevalence of virulence factors such as CagA or VacA of H. pylori isolated from Korean adults compared with those from western countries, the establishment of a mouse model infected with H. pylori isolated from Korean adults is needed to investigate the pathogenesis and to develop vaccines against H. pylori infection in Korea. The aim of this study was to establish the BALB/c mouse model infected with H. pylori isolated from Korean. METHODS: Six-week-old BALB/c mice were inoculated intragastrically with 10(9) CFU of H. pylori. Loss of glandular architecture, erosions and infiltration of inflammatory cells within the lamina propria compared with normal gastric mucosa were scrutinized. Evidence for H. pylori infection was assessed by rapid urease test of gastric mucosa and by microscopic examination using the H & E stain and Warthin-Starry silver stain. RESULTS: Rapid urease test was positive in 55% of all inoculated mice. Definite histologic changes and the evidence of H. pylori colonization were observed in the H. pylori infected group. Significant infiltration of inflammatory cells was observed 6 weeks after the last inoculation and the level of serum IgG against H. pylori was increased from 2 weeks after the last inoculation. CONCLUSIONS: The H. pylori isolated freshly from Korean adults could colonize the stomach of BALB/c mice and induce pathologic alterations that mimics human gastric diseases. This model would facilitate the investigations for the pathogenetic mechanisms of H. pylori infection.
Adult ; Animal ; Base Sequence ; DNA Primers/genetics ; Disease Models, Animal ; Female ; Gastric Mucosa/pathology ; Helicobacter Infections/pathology ; Helicobacter Infections/etiology* ; Helicobacter pylori*/pathogenicity ; Helicobacter pylori*/isolation & purification ; Helicobacter pylori*/genetics ; Human ; Korea ; Mice ; Mice, Inbred BALB C ; Virulence/genetics

OBJECTIVEThis study was aimed to characterize the Helicobacter pylori strains isolated from different geographic regions in China and different ethnic groups in Yunnan province in terms of cagA, iceA, vacA and HP0519 genes which were proposed to be related to the pathogenesis.

METHODS150 Helicobacter pylori strains were collected from Yunnan province, Fujian province and Beijing. Chromosome DNA was extracted and polymerase chain reaction (PCR) was carried out to determine the 3' region of cagA, iceA, vacA and HP0519 status with specific primers. PCR results were analyzed statistically according to their isolated original and clinical outcomes.

RESULTSFor cagA 3' region, 93% (139/150) of the Chinese Helicobacter pylori strains belonged to East Asian type according to the specific primer of TF/JR. Among the 150 strains, 75% (113/150) belonged to iceA1, and 19% (29/150) to iceA2. The dissemination of iceA was not associated with any of the geographic regions, different ethnic groups or different clinical outcomes. 96% (144/150) of the vacA s region belonged to s1. In the vacA middle region, m2, m1b, m1b-m2 were 57% (85/150), 27% (41/150) and 11% (16/150) respectively. However, m1a was only observed in two strains from Fujian. Neither vacA s1 nor m2 showed significant difference between Yunnan, Fujian and Beijing. However, the distribution of mlb-m2 in Yunnan was higher than that in Fujian and Beijing. In Yunnan province, the distribution of vacA s1 was not associated with different ethnic groups but m2 from Bai group was less than other two ethnic groups. The ratio of m1b in Bai group was higher than that in other groups. Both vacA' s region and m region alleles had no significant relationship with the clinical outcomes. With the 15 bp and 24 bp DNA insertion and deletion primers test, 93% (140/150) of the strains were positive. The distributions of the 15 bp and 24 bp DNA insertion or deletion were different according to the different ethnic groups.

CONCLUSIONBy JF/TR primer, 93% of the Chinese strains cagA's 3' region belonged to East Asian type. Most of the Chinese strains vacA's allele was s1. The distribution of vacA s1 had no relationship with the clinical outcome of the isolates. From different geographic regions and ethnic groups, the distribution of vacA m region allele was different. 93% of the Chinese strains HP0519 genes had 24 bp or 15 bp insertion or deletion character. The biological meaning of the polymorphism of HP0519 needs advanced investigation.

China ; Genes, Bacterial ; genetics ; Helicobacter Infections ; ethnology ; genetics ; Helicobacter pylori ; classification ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction

To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.
Antigens, Bacterial/*genetics ; Bacterial Proteins/*genetics ; DNA Fingerprinting/methods ; Gene Amplification/*genetics ; Helicobacter pylori/*genetics ; Helicobacter pylori/isolation & purification ; *Polymorphism, Restriction Fragment Length

Various in situ hybridization (ISH) methods have been used to identify Helicobacter pylori, a causative organism responsible for chronic gastritis and peptic ulcer disease, but they were hard to perform and time consuming. To detect H. pylori in a rapid and easily reproducible way, we developed synthetic biotinylated oligonucleotide probes which complement rRNA of H. pylori. Formalin-fixed and paraffin-embedded tissues from 50 gastric biopsy specimens were examined. Using a serologic test and histochemical stain (Warthin-Starry silver stain and/or Giemsa stain) as a standard, 40 of them were confirmed to be H. pylori-positive. Our ISH was quickly carried out within one hr and results were compared with those obtained from immunohistochemical stain. The ISH produced a positive reaction in 38 of 40 cases (95%). All H. pylori-negative cases failed to demonstrate a positive signal. The ISH has a sensitivity comparable to those of conventional histochemical and immunohistochemical stain, and has high specificity. In conclusion, ISH with a biotinylated oligonucleotide probe provides a useful diagnostic method for detecting H. pylori effectively in routinely processed tissue sections.
Helicobacter Infections/pathology ; Helicobacter Infections/microbiology* ; Helicobacter pylori/isolation & purification* ; Helicobacter pylori/genetics ; Human ; In Situ Hybridization/methods* ; Oligonucleotide Probes ; RNA, Bacterial/analysis ; RNA, Ribosomal, 16S/analysis ; Sensitivity and Specificity ; Silver Staining/methods ; Time Factors

INTRODUCTIONHelicobacter (H.) hepaticus infection causes chronic active hepatitis and induces hepatocellular tumours in A/JCr mice, but evidence of this in humans is scarce. This study aimed to demonstrate the correlation between H. hepaticus and human primary hepatocellular carcinoma (HCC).

METHODSThe sera of 50 patients with primary HCC were tested for the presence of anti-H. pylori and anti-H. hepaticus immunoglobulin G (IgG) antibodies. The liver tissues of patients who tested positive for serum antibody were analysed for H. hepaticus-specific 16S rRNA, H. hepaticus cdtB, H. pylori cagA, H. pylori vacA and H. pylori ureC genes using polymerase chain reaction.

RESULTSAfter the anti-H. pylori antibodies in the serum samples were absorbed by H. pylori antigen, the anti-H. hepaticus IgG serum antibody detection rate was 50.0% in patients with primary HCC. This was significantly higher (p < 0.001) than the detection rate in the benign liver tumour (7.7%) and normal liver tissue (6.3%) groups. Of the 25 primary HCC samples that tested positive for anti-H. hepaticus IgG serum antibody, the H. hepaticus-specific 16S rRNA gene was detected in nine (36.0%) samples. Sequencing showed that the polymerase chain reaction-amplified product exhibited 95.5%-100% homology to the H. hepaticus-specific 16S rRNA gene. Among these nine primary HCC tissue samples, the H. hepaticus cdtB gene was detected in four (44.4%) samples, while no such expression was observed in the benign liver tumour or normal liver tissue groups.

CONCLUSIONThe present study identified the presence of H. hepaticus infection in patients with primary HCC using serological and molecular biological detection, suggesting that H. hepaticus infection may be involved in the progression of HCC.

Adult ; Aged ; Carcinoma, Hepatocellular ; microbiology ; DNA, Bacterial ; genetics ; Female ; Helicobacter Infections ; genetics ; microbiology ; Helicobacter hepaticus ; genetics ; isolation & purification ; Helicobacter pylori ; genetics ; isolation & purification ; Humans ; Immunoglobulin G ; blood ; Liver Neoplasms ; microbiology ; Male ; Middle Aged ; Polymerase Chain Reaction

Discovery of Helicobacter (H.) pylori has led to a fundamental change in our understanding of gastric diseases in humans. Previous studies have found various Helicobacter spp. in dogs and cats, and pets have been questioned as a zoonotic carrier. The present study surveyed the Helicobacter infections and investigated the presence of H. felis and H. pylori infections in domestic and feral cats in Korea. Sixty-four domestic cats and 101 feral cats were selected from an animal shelter. Saliva and feces were evaluated by Helicobacter genus-specific polymerase chain reaction (PCR). Genus-specific PCR positive samples were further evaluated for H. felis and H. pylori using specific primer pairs. Thirty-six of 64 (56.3%) samples from domestic cats and 92 of 101 (91.1%) samples from feral cats were PCR positive; the positive rate of feces samples was higher than that of saliva samples in both groups. H. felis and H. pylori species-specific PCR was uniformly negative. The prevalence of Helicobacter spp. in feral cats was approximately two-fold higher than that of domestic cats. The fecal-oral route may be more a common transmission route not only between cats but also in humans.
Animals ; Cat Diseases/*epidemiology ; Cats ; DNA, Bacterial/genetics ; Feces/microbiology ; Helicobacter Infections/epidemiology/microbiology/*veterinary ; Helicobacter felis/genetics/isolation & purification ; Helicobacter pylori/genetics/isolation & purification ; Korea/epidemiology ; Polymerase Chain Reaction/veterinary ; Saliva/microbiology ; Species Specificity

The polymerase chain reaction was used to develop a method for the detection of Helicobacter pylori, a causative agent of gastritis, as well as for the elucidation of its mode of transmission. A genomic library of Helicobacter pylori DNA in Escherichia coli JM109 was constructed by cloning Hind III-digested DNA fragments into plasmid vector pUC18. The nucleotide sequences from seven recombinant clones were determined and five sets of oligonucleotide primers were synthesized on the basis of the sequences from five clones (B4, B9, B10, C15 and I22). The PCR amplifications with these primers were performed using DNA samples from five strains of Helicobacter pylori, two Campylobacter spp. and eleven species of enteric bacteria. Amplifications of the target DNA fragments in all of 5 strains of Helicobacter pylori were observed from the PCR with primers derived from clone B4, B9, C15 and I22. When the specificity was checked with the DNA samples from 13 other bacteria as template DNA for the PCR, specific amplification that produced the correct size of the target DNA of Helicobacter pylori was shown only in the PCR with primers derived from clone B9 and C15. The detection limit in the PCR amplification, determined by the heat-lysis method, was 500 cells of Helicobacter pylori.
Base Sequence ; DNA, Bacterial/*analysis ; DNA, Recombinant ; Genomic Library ; Helicobacter Infections/diagnosis ; Helicobacter pylori/*genetics/isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Sensitivity and Specificity

The gene encoding urease subunit A (ureA) of Helicobacter pylori (H. pylori) was cloned from H. pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined. Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet. 0.8 kb PCR product was amplified from all H. pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44% G + C content. The DNA sequence was 98% homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino-acid sequences of the ureA were 99% homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H. pylori in the NCBI Entrez database.
Base Sequence ; Cloning, Molecular ; DNA, Bacterial/chemistry ; DNA, Bacterial/genetics ; *Genes, Bacterial ; Genetic Code ; Helicobacter Infections/microbiology ; Helicobacter pylori/enzymology ; Helicobacter pylori/*genetics ; Helicobacter pylori/isolation & purification ; Molecular Sequence Data ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transcription, Genetic ; Urease/*genetics ; Urease/metabolism

To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.
Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; DNA Fingerprinting ; methods ; Gene Amplification ; genetics ; Helicobacter pylori ; genetics ; isolation & purification ; Humans ; Polymorphism, Restriction Fragment Length