Comparison of Next-Generation Sequencing, Real-Time PCR and HRM-PCR for Helicobacter pylori Detection in Pediatric...

In 40 unique pediatric biopsy samples, this study aimed to compare the obtained results of Helicobacter pylori DNA detection using next-generation sequencing (NGS), a real-time PCR-based IVD-certified kit and an established high resolution melting real-time PCR-based method for H. pylori-specific ureA gene. From the same group, the H. pylori DNA was identified in 16 (40.0%) samples in both real-time PCR-based methods, with quantification cycle (Cq) values ranging from 17.51 to 32.21 for the IVD kit. NGS was able to detect H. pylori DNA in 14 (35.0%) samples, with read counts between 7768 and 42,924. While all three methods showed similar detection rates, both PCR variants were slightly more sensitive, identifying H. pylori in two additional samples not detected by NGS. The study highlights the strengths and limitations of each method. NGS, though promising due to its high sensitivity and ability to detect low bacterial load, is still limited by its cost and complexity. Despite these challenges, NGS could complement PCR in diagnosing difficult or ambiguous cases, enabling the detection of multiple pathogens simultaneously. Especially when other infectious etiologies are suspected, NGS could be considered, though PCR variants remain a more attractive and cost-effective option for routine H. pylori detection.