Assessment of real-time PCR for Helicobacter pylori DNA detection in stool with co-infection of intestinal parasites: A comparative study of DNA extraction methods

Abstract

Background: Many studies reported high prevalence of H. pylori infection among patients co-infected with intestinal parasites. Molecular approach for the DNA detection of those microbes in stool have been proposed. However there are a few reports that evaluated the effect of bead-beating in relation to the H. pylori outcome. Therefore, we developed and evaluated two TaqMan-based real-time PCR (rt-PCR) qualitative assays for the detection of ureC (glmM) and cagA of Helicobacter pylori on DNA extracted by three procedures. Results: The two PCRs were analysed on 100 stool samples from patients who were screened for intestinal parasites. Three DNA extraction procedures were used: 1) automation with bead beating, 2) automation without bead beating and 3) hand column. The specificity of the new assays was confirmed by sequencing the PCR products and by the lack of cross-reactivity with other bacteria or pathogens DNA. Rt-PCR assays showed a detection limit of 10^4 bacteria/200 mg stool. The ureC_PCR with bead beating process was compared to conventional stool antigen test (SAT), with 94.12 and 93.75% of respectively sensitivity and specificity. However, the discordant samples were confirmed by DNA sequencing suggesting a potential higher sensitivity and specificity of PCR. Conclusions: Our findings showed that the automation with bead-beating -suggested procedure for intestinal parasitic infections- can reach highly sensitive results in H. pylori detection on stool compared also with SAT. Thus, this work can provide new insights into the practice of a clinical microbiology laboratory in order to optimize detection of gastro-intestinal infections. Further studies are needed to better define the clinical value of this technique.