Helicobacter pylori 23S rRNA Gene Sequencing for the Identification of Mutations Associated with Clarithromycin Resistance in FFPE Samples
Midori Mitui, Shama K Khokhar, Nora K Leos, Christopher D Doern, Jason Y Park. Children's Medical Center, Dallas, TX; UT Southwestern Medical Center, Dallas, TX
Background: Helicobacter pylori (H. pylori) causes gastritis, gastric ulceration, and gastric carcinoma. The antibiotic clarithromycin (CLAR) is commonly used to treat infections with this organism; however, some populations have a high prevalence of CLAR resistance. Mutations in the 23S rRNA gene of H. pylori mediate resistance. Recently, clinical guidelines have recommended testing of 23S rRNA for the management of persistent H. pylori infection. Clinical testing for 23S rRNA in the United States is not widely available. We designed an assay based on PCR amplification and sequencing of the H. pylori 23S rRNA gene.
Design: H. pylori 23S rRNA testing was targeted to a DNA region that contains mutations associated with CLAR resistance. H. pylori sequences from GenBank were used to design H. pylori specific primers; however, the true specificity could not be determined because of unavailable sequence information from non-pylori Helicobacter species. Cultured H. pylori DNA was obtained (ATCC) for optimization of the PCR reaction. Β-actin PCR was performed as internal control. 46 FFPE archival samples from 33 cases were evaluated. These samples had been previously evaluated by H. pylori immunohistochemistry (IHC). H. pylori load in each FFPE sample was assessed by counting all organisms present on a single IHC slide tissue level.
Results: All FFPE tissues with ≥48 organisms per IHC tissue were detected by PCR and sequencing. By sequencing, 63% of cases had the known resistance mutation (2143A>G) in the heterozygous or homozygous state. A nucleotide substitution (2131C>T) of unknown significance was identified in 1 case. In 3 FFPE samples with IHC organisms counts per tissue level of ≤23, there was no H. pylori PCR amplification. Furthermore, we examined 5 samples from 4 cases of H. heilmannii. Only 2 samples had ≥48 organisms per tissue level and these were both positive for H. pylori 23S rRNA PCR; sequencing for mutation evaluation is pending.
Conclusions: In this ongoing study, the CLAR resistance mutation (2143A>G) was identified in 63% of H. pylori cases. Indeed, recent guidelines consider >20% to be a high resistance rate requiring the use of non-CLAR regimens. This DNA sequencing test can be implemented for the clinical evaluation of single cases or for epidemiologic studies to determine the prevalence in various communities. Future studies will examine the specificity of this assay for H. pylori versus other Helicobacter species such as H. heilmannii.
Tuesday, March 5, 2013 2:05 PM
Proffered Papers: Section H2, Tuesday Afternoon