Detection of Helicobacter pylori in stool samples of young children using real-time polymerase chain reaction.

Abstract

The aims of this study were to develop and validate a multiplex real-time polymerase chain reaction (q-PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H.pylori-positive samples. Archived stool samples from 188 children aged 6-9years and 272 samples of 92 infants aged 2-18months were tested for H.pylori antigens using enzyme immunoassay (EIA). A multiplex q-PCR assay was designed to detect H.pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q-PCR and EIA. Laboratory validation of the q-PCR assay using quantitated H.pylori ATCC 43504 extracted DNA showed S-shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross-reactivity with other bacterial pathogens was noted. Applying the multiplex q-PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H.pylori infection was 50% (95% CI 43%-57%) by q-PCR (urease cycle threshold <44) vs 59% (95% CI 52%-66%) by EIA. Kappa coefficient was .80 (P<.001) and .44 (P<.001) for children aged 6-9 years and 2-18months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing. The developed q-PCR can be used as a cotechnique to enhance the accuracy of H.pylori detection in epidemiological studies and in clinical settings.