| The evidences from clinical and epidemiological studies show that the Helicobacter pylori has been regarded as the main cause of chrinic gastritis and peptic ulcer, plays an important role in the pathogenesis of MALT lymphoma and gastric cancer. The rate of multitude infection of H.pylori is more than 50% in the whole world . Eradication of H.pylori not only aids ulcer healing and prevents ulcer recurrence but also reduces the risk of gastric carcinoma. Therefore eradication therapy for H.pylori has developed rapidly in recent years, The use of antibiotics has become more widespreader in the eradication of this pathogen. Clarithromycin is the most effective antibiotic in eradication of H.pylori. The most important and advocated therapy, triple therapy, often includes clarithromycin. Study indicated that the triple therapy, which includes clarithromycin, can improve effectiveness by an average of 10%~20%. Although this advanced triple therapy,includes clarithromycin, can assure rapid symptom relief, improve ulcer healing, but treatment failure still occurs in some cases. Antibiotic resistance are currently regarded as the major causes of eradication failure. The study indicated that 5%~20% patients were resistant to clarithromycin . Clarithromycin resistsnce is one of the major causes of eradication failure. Research find that clarithromycin resistsnce reduce effectiveness by an average of 55%. There is a clinical need for rapid and accurate diagnostic methods to determine clarithromycin resistsnce . The conventional method to determine clarithromycin resistsnce of H.pylori is based on the analysis of cultured strains by agar diffusion or dilution or the epsilometer (E-test). But H.pylori is a relatively fastidious and slow growing microaerophilic microorganism . It takes at least 10~14 days from initial receipt of the gastric biopsy to reporting the sensitivity results. Molecular assay for detecting clarithromycin resistsnce in H.pylor is a rapid and accurate method. Previous findings have demonstrated that clarithromycin resistsnce is due to a single point mutation in the 23S rRNA. The mutation described have been associated with specific restriction sites, namely BsaI(A2143G), BbsI(A2142G). Our investigation aim to developed a nested PCR system which amplifies a segment of the 23S rRNA gene from gastrict biopsies and stool samples, so that Helicobacter pylori infection and the mutation associated with clarithromycin resistsnce can be examined simultaneously. Objective:To establish a nested PCR system for the detection of Helicobacter pylori 23S rRNA gene in gastrict biopsies based diagnosis of Helicobacter pylori infection. Methods: Fifty seven gastric biopsies, obtained through upper gastrointestinal endoscopy from 57 patients (38 men, mean age 44 years, 21gastritis, 30 peptic ulcer ,and 6 gastric cancer), were processed from december 2003 to july 2004. The H. pylori status was evaluated based on three different tests ( rapid urease test,histology and 14C-urea breath test) ,and defined as positive when two of three tests were positive, negative when two of three tests were negative. DNA from 57 gastric biopsies ,42 H.pylori positive and 15 H.pylori negative, were all amplified by not only Fluorescence 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. Results: The Fluorescence PCR method correctly classified 38 of 42 H.pylori-positive patients and 13 of 15 H.pylori-negative patients (sensitivity 90.5%, specificity 86.7%, PPV 95.0%, NPV 76.5%, accuracy89.5%). The nested PCR method correctly classified 35 of 42 H.pylori-positive patients and 14 of 15 H.pylori-negative patients (sensitivity 85.7%, specificity 93.3%, PPV 97.3%, NPV 70%, accuracy 87.7%). But the difference with the Fluorescence PCR was no significance. Restriction analysis of 37 nested PCR products show that foursamples contained clarithromycin resistsnce strains due to the presence of mutation at position 2143, Whereas no PCR p...
|
PDF Full Text Request