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

Area of Science:

• Microbiology
• Molecular Biology
• Pediatric Infectious Diseases

Background:

• Helicobacter pylori is a significant pathogen in pediatric populations.
• Accurate detection of H. pylori in stool is crucial for epidemiological studies and clinical management.
• Understanding antibiotic resistance patterns, such as clarithromycin resistance, is essential for effective treatment.

Purpose of the Study:

• To develop and validate a multiplex real-time polymerase chain reaction (q-PCR) assay for detecting Helicobacter pylori in children's stool samples.
• To determine the prevalence of H. pylori infection in a pediatric cohort.
• To assess the prevalence of clarithromycin resistance and the cagA gene in H. pylori-positive samples.

Main Methods:

• A multiplex q-PCR assay was designed to detect H. pylori 16S rRNA and urease genes, with human RNase P as an internal control.
• Stool samples from children (6-9 years) and infants (2-18 months) were tested using the developed q-PCR and enzyme immunoassay (EIA).
• Agreement between q-PCR and EIA was assessed using the Kappa coefficient. Sequencing confirmed clarithromycin resistance mutations and cagA gene presence.

Main Results:

• The validated q-PCR assay demonstrated a limit of detection of 1 CFU/reaction with no cross-reactivity.
• H. pylori prevalence was 50% by q-PCR and 59% by EIA, with good agreement (Kappa = 0.80) in older children.
• Sixteen samples were positive for the cagA gene, and three harbored the clarithromycin resistance mutation (A2143G).

Conclusions:

• The developed multiplex q-PCR assay is a reliable tool for H. pylori detection in pediatric stool samples.
• This assay can enhance the accuracy of H. pylori detection in both epidemiological and clinical settings.
• The study provides valuable data on H. pylori prevalence and antibiotic resistance in young populations.