Clinical validation of a fecal PCR assay for Helicobacter pylori detection: multicenter comparative study with urea breath test and commercial molecular diagnostics

ABSTRACT Helicobacter pylori infection is linked to gastritis, peptic ulcers, and gastric cancer. The urea breath test (UBT), while gold standard for active infection diagnosis, poses radioactive risks and equipment dependency. Fecal molecular detection offers a non-invasive alternative but requires validation. This multicenter study evaluates a novel fecal PCR assay (Cellrun PCR assay, 16S rRNA-targeted) against UBT and a commercial kit (comparison reagents). A multicenter trial enrolled 866 patients (May 2023–Sep 2024) from four hospitals. Of 777 valid samples, positive detection rates, diagnostic consistency (kappa), and clinical performance (sensitivity/specificity) were compared across UBT, Cellrun PCR assay, and comparison reagents. Positive rates were 44.02% (UBT), 43.89% (Cellrun PCR assay), and 44.14% (comparison reagents), with no significant differences ( P > 0.05). Cellrun PCR assay exhibited near-perfect agreement with UBT (Kappa = 0.9713, P < 0.001) and matched comparison reagents’ consistency (Kappa = 0.966, P < 0.001). Sensitivity and specificity for Cellrun PCR assay were 98.2% (AUC = 0.985), comparable to comparison reagents (AUC = 0.983; P = 0.614). Weak-positive agreement was 85.7%, while post-treatment sensitivity/specificity reached 94.74%/97.06% (AUC = 0.985). Cellrun PCR assay achieved 100% accuracy in interference samples (Kappa = 1.0). False negatives correlated with low bacterial loads or processing variability; false positives arose near detection thresholds. Cellrun PCR assay demonstrates UBT-comparable sensitivity and specificity, validating its equivalence to established molecular diagnostics for Helicobacter pylori detection. IMPORTANCE Helicobacter pylori infection is a major global health burden, linked to gastritis, peptic ulcers, and gastric cancer. Current gold-standard urea breath tests (UBTs) carry radioactive risks and require specialized equipment, limiting accessibility. Fecal molecular detection offers a non-invasive alternative, but existing assays need robust validation. Our study validates the novel Cellrun PCR assay, showing it matches UBT’s accuracy and established commercial kits. This user-friendly, non-radioactive tool enhances diagnostic accessibility, especially in resource-limited settings, enabling early H. pylori detection and targeted treatment—critical for reducing associated morbidity and mortality. Its reliability supports broader clinical adoption, advancing global efforts to manage this prevalent infection.