Clinical Value Of Assaying Helicobacter Pylori 23SrRNA Gene Mutant In Gastric Mucosa And Dental Plaques For The Diagnosis Of Clarithromycin Resistance

Helicobacter pylori (H. pylori) is a spiral-shaped Gram- negative flagellate bacterium that has been implicated as a major human gastric pathogen responsible for chronic gastritis, peptic ulcer diseases, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. An estimated 50% individuals are actively infected with H. pylori. It is generally accepted that treatment to eradicate Helicobacter pylori in patients with proved peptic ulcer is cost effective and benefits the patient and society.Antibiotic resistance is an increasing problem for the treatment of infectious diseases. Much publicity is currently given to the widespread use of antibiotics and the threats posed by the emergence of pathogenic organisms resistant to all available antibiotics.Molecular test methods might have an impact on improving the availability and accuracy of information on H pylori antimicrobial resistance to guide in the selection of primary as well as secondary backup treatment regimens.Most of the current regimens for the eradication of H. pylori include clarithromycin (i.e. triple therapy with a proton pump inhibitor [PPI], amoxicillin and clarithromycin or that with a PPI, metronidazole and clarithromycin). Bacterial susceptibility to clarithromycin is significantly related to eradication rates of H. pylori by clarithromycin-based therapy.Bacterial resistance of H. pylori to clarithromycin is related to the structural change of 23S rRNA. Because the target of clarithromycin is 23S rRNA, the structural change of 23S rRNA results in resistance to clarithromycin. This structural change of 23S rRNA is caused by the single nucleotide polymorphism (SNP) of the 23S rRNA gene, at the position of 2142 or 2143 in most cases.Therefore, bacterial resistance to clarithromycin can be determined by a genetic test. Development of an inexpensive and reliable high-throughput method for scoring of such a SNP is imperative in H. pylori eradication therapy with a regimen including clarithromycin.The allele specific primer-polymerase chain reaction (ASP-PCR) method is one of the tools used to determine SNPs with a high specificity and sensitivity from genomic or complementary DNA samples. It can measure the SNPs easily within a short period by PCR amplification alone without digestion with restriction enzymes or direct sequencing. In the ASP-PCR analysis, PCR amplification is performed by the specific primer whose second base from the 3′end is designed to match the site of the SNP and the third base is designed to have the mismatch sequence in order to yield allele-specific PCR amplification.The SNPs can be determined by whether or not the PCR amplicons corresponding to the specific primers are observed.Molecular assays for detecting clarithromycin resistance in H pylori are all based on detection of mutations in the 23SrRNA genes. In particular, the main 23SrRNA mutations are an adenine-to-guanine transition at positions 2142 and 2143. These single point mutations also generate specific restriction sites, namely BsaI and BbsI, which can be used for the rapid screening of clarithromycin resistance.The oral cavity is a reservoir for H.pylori colonization and may imply that there is a risk of the relapses of gastric and duodenal H.pylori infection.Development of an inexpensive and reliable high-throughput method frome dental plaques is imperative in H. pylori eradication therapy with a regimen including clarithromycin.Objective:The aim of the present study was to develop the ASP-PCR assay for determining SNPs at positions 2142 and 2143 of the 23SrRNA gene of H. pylori from gastric biopsies. To establish a nested PCR system for the detection of Helicobacter pylori 23S rRNA gene in dental plaques based diagnosis of Helicobacter pylori infection.Methods:1 The study subjects consisted of 83 gastric biopsies, obtained through upper gastrointestinal endoscopy from 83 patients (49 men, median age of 47 years, 16 gastritis, 60 peptic ulcer , and 7 gastric cancer), were processed from June 2006 to November 2006. The H. pylori status was evaluated based on three different tests ( rapid urease test,histology and 14C-urea breath test) ,and was defined as positive when two of three tests were positive, negative when two of three tests were negative. DNA from 83 gastric biopsies ,65 H. pylori positive and 18 H. pylori negative, were all amplified by not only ASP-PCR but also nested PCR, the PCR products of the latter were subsequently digested with BsaI and BbsI.. Statistical analysis was performed by chisquare test with SPSS 12.0.2 45 patients with H. pylori-positive confirmed by both 14C-urea breath test and rapid urease test were enrolled, including of 23 patients with chronic superficial gastritis, 22 with duodenal ulcer. DNA from 45 dental plaques were all amplified by nested PCR. The products were digested by BsaI and BbsI.Results:1 Fifty eight of 65 patients were infected with wild-type strains of H. pylori.One patients were infected with strains with A2143G mutations. Six patients were infected with both wild-type strains and those with A2143G mutations. For these strains , contained clarithromycin resistsnce strains,the presence of the mutation was confirmed by sequence analysis. accuracy 100%.The nested PCR method correctly classified 56 of 65 H. pylori-positive patients and 17 of 18 H. pylori-negative patients. Restriction analysis of 48 nested PCR products show that seven samples contained clarithromycin resistsnce strains due to the presence of mutation at position 2143, Whereas no PCR products contained mutation at position 2142. For these strains , contained clarithromycin resistsnce strains,the presence of the mutation was confirmed by sequence analysis.2 Of 25 dental plaques specimens amplication was successful in 45(55.6%) in the first PCR,and 28 samples were amplified successfully by nested PCR(62.6%). Restriction analysis of 28 nested PCR products show that one samples contained clarithromycin resistsnce strains due to the presence of mutation at position 2143, Whereas no PCR products contained mutation at position 2142. For these strains , contained clarithromycin resistsnce strains,the presence of the mutation was confirmed by sequence analysis.Conclusion:1 The ASP-PCR method for the determination of SNPs of the H. pylori 23S rRNA of H. pylori (from adenine to guanine mutation at the positions of 2142 and 2143) from the genomic DNA extracted from gastric mucosal samples is a useful method to detect clarithromycin-resistant strains of H. pylori easily, without digestion with restriction enzymes or direct sequencing.2 Nested PCR system, which amplifies a 425bp segment of 23S rRNA gene containing the mutation points with primers specific for H. pylori ,can detect H. pylori infection in dental plaques and the mutation associated with clarithromycin resistsnce simultaneously.