Detection of point mutations on 23S rRNA of Helicobacter pylori and resistance to clarithromycin by PCR-RFLP in Spain
Abstract number: P1865
Agudo S., Alarcón T., Pérez-Pérez G., López-Brea M.
Objective: The identification and characterization of clarithromycin resistant Helicobacter pylori strains is a long and expensive process that requires culture and isolation of H. pylori from gastric biopsies. The purpose of the present study was to identify H. pylori strains resistant to clarithromycin and determine the most common point mutation present in the 23S rRNA gene using a PCR-RFLP technique.
Methods: Gastric biopsies were obtained by endoscopy from paediatric patients with gastric symptoms. H. pylori was cultured according to standard microbiological procedures and clarithromycin resistance determined by E-test. DNA extraction was performed by NucliSens platform with the NucliSens magnetic extraction reagents (bioMérieux) according to manufacturers instructions. Clarithromycin resistant and sensitive strains were determined by PCR and DNA sequencing for mutations in 23S rRNA gene. Restriction fragment length polymorphism was performed using BsaI enzyme to detect restriction sites that correspond to the mutation.
Results: We found 42 out of 118 (35.6%) strains resistant to clarithromycin by E-test. E-test results were confirmed for the presence of point mutation in 34 (88.1%) of these strains. There were 8 H. pylori strains resistant to clarithormycin by E-test but without any point mutation in the 23 rRNA gene. Mutation A2143G was found in 85.3% of the strains. Restriction enzyme analyses with BsaI was able to detect the presence of that mutation in 100% of the resistant cases.
Conclusion: PCR-RFLP is an usefull method to detect clarithromycin-resistant H. pylori infection in countries with a high prevalence as Spain. Only A2143G mutation could be detected with this assay, but it is the most prevalent in Spain as well as in the other countries. It may be useful to apply this methodology before choosing regimens of H. pylori eradication.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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