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Updated: Aug 17, 2026
Detection of Helicobacter pylori Infection and Antibiotic Resistance via Stool Quantitative Polymerase Chain Reaction Analysis
Published on: May 16, 2025
Polymerase chain reaction--restriction fragment length polymorphism analysis of clarithromycin-resistant Helicobacter
Mina Booka1, Masumi Okuda, Kouichirou Shin
1Department of Pediatrics, Wakayama Medical University, Wakayama, Japan.
Insights
A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using stool samples can rapidly detect clarithromycin-resistant Helicobacter pylori infection in children. This noninvasive technique aids in selecting effective H. pylori eradication treatments.
Area of Science:
- Microbiology
- Pediatric Infectious Diseases
- Molecular Diagnostics
Background:
- Helicobacter pylori infection is a significant global health concern, particularly in children.
- Antibiotic resistance, especially to clarithromycin, complicates eradication therapy.
- Noninvasive diagnostic methods are crucial for pediatric patient management.
Purpose of the Study:
- To develop and validate a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for detecting clarithromycin-resistant H. pylori in pediatric stool samples.
- To assess the prevalence of clarithromycin resistance in H. pylori infections among children.
Main Methods:
- A novel PCR-RFLP method was established targeting the 23S rRNA gene of H. pylori in stool DNA.
- Restriction enzymes MboII, BceAI, and BsaI were used to identify specific resistance mutations (A2142G, A2142C, A2143G).
- The assay was validated against the H. pylori stool antigen test (HpSA) in a cohort of 23 children.
Main Results:
- The PCR-RFLP assay demonstrated 89% sensitivity, 100% specificity, and 91% accuracy compared to HpSA.
- Out of 16 H. pylori-positive samples, 31% showed resistance to clarithromycin based on enzyme digestion patterns.
- The method successfully identified clarithromycin resistance mutations in pediatric stool samples.
Conclusions:
- PCR-RFLP using stool samples is a rapid, reliable, and noninvasive method for detecting clarithromycin-resistant H. pylori in children.
- This diagnostic approach can guide the selection of appropriate H. pylori eradication regimens.
- The findings highlight the utility of molecular methods in pediatric infectious disease diagnostics.
Background:
To analyze clarithromycin-resistant Helicobacter pylori infection in children, we developed a method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using stool samples.
Materials And Methods:
Twenty-three children without significant upper abdominal symptoms were included (mean age 7.0 years). Of these, 18 and five were diagnosed as H. pylori-positive and -negative, respectively, by the H. pylori stool antigen test (HpSA). The DNA from the stool samples was purified using the QIAamp DNA Stool Minikit (QIAGEN). The PCR was performed on the purified DNA using oligonucleotide primers designed to amplify the 23S rRNA gene of H. pylori. The PCR products were reacted with restriction enzymes MboII, BceAI, and BsaI to detect mutations A2142G, A2142C, and A2143G, respectively.
Results:
Sixteen of the 18 HpSA-positive samples were PCR-positive, and all five HpSA-negative samples were PCR-negative. Thus, the PCR had 89% sensitivity and 100% specificity, with 91% accuracy in reference to HpSA. Of the 16 PCR-positive samples, one and four were digested with MboII and BsaI, respectively, indicating 31% prevalence of CAM-resistance.
Conclusions:
We conclude that the PCR-RFLP using stool samples is a rapid and reliable method to noninvasively detect clarithromycin-resistant H. pylori infection in children. It may be useful before choosing regimens of H. pylori eradication.
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