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

Abstract

Background

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.

Materials and methods

Archived stool samples from 188 children aged 6-9 years and 272 samples of 92 infants aged 2-18 months 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.

Results

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 2-18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing.

Conclusions

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.

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