Genotyping Study On Helicobacter Pylori Isolated From Patients Of Chronic Gastritis And Peptic Ulcer In Henan

Helicobacter pylori (H.pylori) infection, one of the most common bacterial infections of the human being, has been identified as a global question. Many studies showed that this organism is the main pathogenic agent of peptic ulcer and chronic gastritis. And even more, it has close relationships with gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma according to recent reports. In 1994, WHO classified H.pylori as a definite (class I ) carcinogen. Center for Diseases Control and Prevention of the United States has declared H.pylori infectious diseases as one of the new emerging infectious diseases.However, many epidemiological research did not show that all H.pylori infected individuals would have the gastrointestinal diseases. 20-30% of the infected persons are asymptomatic, 50% may have gastritis in varying degrees, 10-15% may get peptic ulcer; a small part might develop gastric cancer. Thus it was deduced that only some H.pylori was related to gastrointestinal diseases and it was possible that H.pylori of different genotypes may cause differrent diseases. The mechanism of pathogenic diversity and the mode of transmission are not clear at present. The poverty of knowledge in these areas is in part due to the lack of a reliable universal classification method. That is why this study aims to typing H.pylori by their genetic features. Many studies have suggested that a high degree of inter-strain genomic heterogeneity is a characteristic of H.pylori. therefore, RAPD and PCR techniques were used to classify 92 clinical isolates of H.pylori and to explore the relationship between the H.pylori genotypes and different diseases in this study.Subjects and Methods1. H.pylori group Classification: H.pylori isolated from chronic gastritis patients were classified into chronic gastritis group (CG). Those from gastric ulcer patients were gastric ulcer group (GU). Others were no-patients of CG/PU group (NP).2. Isolation and identification of H.pylori: H.pylori isolates were seperated from gasric mucosa and gastric juice. Gastric mucosal biopsy specimens were obtained through routine endoscopy. Gastric juice were gathered by gastric canal from inpatients who neeeded operation. Specimens of gastric mucosa and gastric juice were cultured on Improved-Bush-Culture-Base. Suspected colonies which exhibited typical colonial morphology, if testified as Gram stainning negative and chemical reaction positive, were subcultured. All isolates were kept in storing liquids at -80°C.3. Random amplified polymorphism DNA (RAPD) analysis: H.pylori genome DNA were random amplified by the following short primer, 5'-CCGCAGCCAA-3'. RAPD production were separated, observed and recorded under the help of gel electrophoresis and gel picture system. All the bands were coded and analyzed by SPSS 13.0.4. PCR analysis of vacA, cagA, cagE, cagT: PCR amplify vacA, cagA, cagE, cagT gene fragment to find out their situation of presence or absence in H.pylori isolates of different groups. The bands of PCR pattern were coded and analyzed by SPSS13.0 too.5. Synthetic cluster analysis: In order to make synthetic cluster analysis, the result of RAPD and PCR analysis mentioned above were integrated into one database. Combined result based on this database was obtained through cluster analysis. Categories obtained by this method were considered as different genotypes. 6. VacA toxicity detection: vacuolization cytotoxin was extracted and its toxicity was detected by Hela cell under the condition of 5% CO2, 37°C, 24hrs.Results1. The result of RAPD showed that 92 H.pylori isolates had different genetic fingerprints. That is to say that the genome of H.pylori has the character of high diversity. SPSS13.0 classified all isolates into 4 genotypes by cluster analysis. But distribution of these genotypes showed no statistical significant correlation among CG, GU and NP at genome level.2. PCR analysis indicated that all isolates contained vacA gene, while some isolates lost one or more of the other three (cagA, cagE and cagT). The typing result of these three genes did not show the real relationship between genotypes of H.pylori and different gastrointestinal diseases (CG, GU and NP).3. The combined result of RAPD and cagA, cagE and cagT could classify 92 H.pylori isolates into four genotypes. These four genotypes of H.pylori had different proportion in different disease groups (CG, GU and NP). It indicated that different genotypes of H.pylori had relationship with different diseases in the multiplex gene level. So it is necessary to build a practical molecular typing system for H.pylori. Furthermore, the results also showed that the numerical classification of the multiple gene is better than typing method of single gene. RAPD and PCR product typing method are simple and convenient and need a little template, so they possess cheerful prospect in gene typing for H.pylori.4. Fifty percent (46/92) of all the isolates expressed VacA toxin (tox~+) detected by Hela cell. However, the distribution of tox~+ isolates among different diseases or genotypes did not appear any statistical relationship.ConclusionsThis case-control study detected gene characters of 92 H.pylori isolates by RAPD and PCR technology, and classified them into different genotypes through statistical cluster analysis. Conclusions are as followed. 1. RAPD fingerprints were different from each other, so it can be used to identify H.pylori. However, it did not show that H.pylori of different genotype classified based on RAPD alone have relationship with gastrointestinal diseases in this study.2. The result of PCR of the four genes (vacA, cagA, cagE and cagT) indicated that the typing method of one single gene did not show the real relationship between H.pylori of different genotype and different gastrointestinal diseases.3. Synthetic cluster analysis of RAPD and PCR classified 92 H.pylori into 4 genotypes with statistical significant distribution difference among CG, PU and NP groups. It provided a good multiplex gene typing method to explore the relationship between H.pylori and gastrointestinal diseases.4. VacA toxicity expression did not effectively typing H.pylori. It showed no statistical correlation with either H.pylori genotypes or different diseases.