Enhancing pathogen detection in pediatric diarrheal stools: The case of molecular multiplexing with a real-time PCR assay
- 1ICMR-National Institute for Research in Bacterial Infections, Kolkata, India
Abstract
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. Author summary: Diarrhea remains a leading cause of childhood death in low-resource settings. Less sensitive conventional diagnostics methods often fail to identify the diarrheal pathogens. We compared a multiplex real-time PCR (GastroFinder 2SMART) assay with standard laboratory methods using stool specimens collected from 153 children with diarrhea. Using conventional diagnostic accuracy metrics and Bayesian latent class analysis—which estimates true test performance without assuming a perfect gold standard—we found that real-time PCR detected pathogens in 81% of samples versus 43.1% by conventional methods (p < 0.001). Detection improved substantially for bacteria (61% vs. 12%) and viruses (67% vs. 27%). Mixed infections were more common by the real-time PCR (75.8% vs. 12.1%). Performance varied by pathogen type: Shigella/EIEC showed excellent discriminatory ability (Youden Index 0.88; sensitivity 93.2%, specificity 95.4%), and negative predictive values exceeded 95% for most pathogens, indicating the use of GastroFinder 2SMART as a screening tool. However, rotavirus sensitivity was unexpectedly low (15%), a critical limitation for pediatric populations. These findings support the integration of molecular methods into routine diarrheal testing to improve pathogen detection and also highlight the need for additional rotavirus testing and careful pathogen-specific interpretation of results.
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BibTeX
@article{chowdhury2026enhancing,
title = {{Enhancing pathogen detection in pediatric diarrheal stools: The case of molecular multiplexing with a real-time PCR assay}},
author = {Goutam Chowdhury and Melissa Glenda Lewis and Alok Deb and Mamta Chawla-Sarkar and Sandipan Ganguly and Paulami Dutta and Santasabuj Das and Asish K. Mukhopadhyay and Thandavarayan Ramamurthy},
journal = {PLOS Neglected Tropical Diseases},
year = {2026},
month = nov,
doi = {10.1371/journal.pntd.0014823},
url = {https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014823},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Enhancing pathogen detection in pediatric diarrheal stools: The case of molecular multiplexing with a real-time PCR assay
AU - Goutam Chowdhury
AU - Melissa Glenda Lewis
AU - Alok Deb
AU - Mamta Chawla-Sarkar
AU - Sandipan Ganguly
AU - Paulami Dutta
AU - Santasabuj Das
AU - Asish K. Mukhopadhyay
AU - Thandavarayan Ramamurthy
PY - 2026
DA - 2026/11/02
JO - PLOS Neglected Tropical Diseases
DO - 10.1371/journal.pntd.0014823
UR - https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014823
AB - 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. Author summary: Diarrhea remains a leading cause of childhood death in low-resource settings. Less sensitive conventional diagnostics methods often fail to identify the diarrheal pathogens. We compared a multiplex real-time PCR (GastroFinder 2SMART) assay with standard laboratory methods using stool specimens collected from 153 children with diarrhea. Using conventional diagnostic accuracy metrics and Bayesian latent class analysis—which estimates true test performance without assuming a perfect gold standard—we found that real-time PCR detected pathogens in 81% of samples versus 43.1% by conventional methods (p < 0.001). Detection improved substantially for bacteria (61% vs. 12%) and viruses (67% vs. 27%). Mixed infections were more common by the real-time PCR (75.8% vs. 12.1%). Performance varied by pathogen type: Shigella/EIEC showed excellent discriminatory ability (Youden Index 0.88; sensitivity 93.2%, specificity 95.4%), and negative predictive values exceeded 95% for most pathogens, indicating the use of GastroFinder 2SMART as a screening tool. However, rotavirus sensitivity was unexpectedly low (15%), a critical limitation for pediatric populations. These findings support the integration of molecular methods into routine diarrheal testing to improve pathogen detection and also highlight the need for additional rotavirus testing and careful pathogen-specific interpretation of results.
SN - 1935-2735
ER -