Enhancing pathogen detection in pediatric diarrheal stools: The case of molecular multiplexing with a real-time PCR assay

Diarrhea is a leading cause of pediatric morbidity and mortality worldwide, frequently caused by various bacterial, viral, and protozoan pathogens. Identification of these pathogens is important in clinical settings for proper management of the infection. Conventional stool culture technique is time-consuming for the detection of diarrheagenic pathogens. In addition, these traditional procedures lack sensitivity, require a specific laboratory structure, and well-trained staff. Conversely, multiplex molecular detection methods offer rapid results with less hands-on time and more sensitivity. This study compared the diagnostic performance of conventional microbiological methods and GastroFinder 2SMART multiplexed real-time PCR (real-time PCR) assay for detecting enteric pathogens in pediatric stool samples. Overall pathogen detection was significantly higher using this real-time PCR (81%) compared to the conventional assays (43.1%), reflecting differences in both sensitivity and pathogen panel breadth. Rotavirus, adenovirus, Shigella , and Giardia lamblia were frequently identified by conventional methods, whereas real-time PCR commonly detected adenovirus, Campylobacter, EPEC, ETEC, and norovirus. Certain pathogens such as Yersinia enterocolitica , Clostridioides difficile , STEC, and Dientamoeba fragilis were not tested in the conventional panel and therefore excluded for comparison. Sensitivity estimates varied, with lower sensitivity observed for EPEC, Cryptosporidium, G. lamblia , and rotavirus, partly due to the small sample numbers and wide confidence intervals. Specificity exceeded 90% for most pathogens, and diagnostic accuracy was > 90% for Shigella /EIEC, Salmonella, Cryptosporidium , Giardia lamblia , and astrovirus. Bayesian latent class analysis demonstrated the highest sensitivity for Shigella /EIEC (93.2%), followed by norovirus and adenovirus, while overall test performance, measured by the Youden Index, was greatest for Shigella /EIEC. These findings highlight the diagnostic use of real-time PCR and support its integration into routine diarrheal diagnostics to improve pathogen detection and clinical management.