Evaluation of multiplex ARMS-PCR for detection of Helicobacter pylori mutations conferring resistance to clarithromycin and levofloxacin
Abstract
Helicobacter pylori bacterium is a major cause of gastritis. With increasing use of antibiotics to treat infections, mutation resistant strains have emerged in most human populations. To effectively treat patients to help resolve infections, the clinician needs information on the antibiotic susceptibility profile of the infection. Therefore, a rapid and accurate test is required to provide this information. To address this issue, we designed and validated a real time multiplex ARMS-PCR assay for rapid detection of highly prevalent H. pylori clarithromycin and levofloxacin resistance mutations. The aim of the study was to evaluate the analytical and diagnostic sensitivity and specificity of ARMS-PCR, using direct Sanger sequencing of the known resistance mutations as the gold standard. In preliminary studies using a defined number of plasmids with clarithromycin and levofloxacin resistance mutations, the analytical sensitivity of our ARMS-PCR assay was 50 plasmid copies, equating to around 50 bacterium in a gastric biopsy sample. In terms of specificity, the assay was highly specific for the targeted resistance mutations. The assay was also able to reliably and efficiently detect heteroresistance of clarithromycin and levofloxacin mutations, even at a disproportional ratio of 1:1000. From the analysis of 192 samples with suspected H. pylori infections, the diagnostic sensitivity and specificity of the assay was very high for detection of clarithromycin resistance (100% and…
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