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Published on: March 10, 2020
Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples
U Schotte1, T Hoffmann2, N G Schwarz3
1Department A - Veterinary Medicine, Central Institute of the Bundeswehr Medical Service Kiel, Kronshagen, Germany.
Insights
Storing stool samples on Whatman papers or flocked swabs is effective for real-time PCR pathogen detection in tropical regions. This method maintains detection frequencies comparable to direct stool nucleic acid extraction.
Area of Science:
- Tropical medicine
- Molecular diagnostics
- Infectious disease epidemiology
Background:
- Accurate pathogen detection is crucial for diagnosing gastrointestinal illnesses in children.
- Traditional stool sample preservation and transport can be challenging in tropical environments.
- Exploring alternative sample preservation methods is vital for improving diagnostic accessibility.
Purpose of the Study:
- To compare the efficacy of real-time PCR for detecting gastrointestinal pathogens using nucleic acid extraction from stool samples versus samples stored on Whatman papers or flocked swabs at ambient tropical temperatures.
- To assess the utility of cycle threshold (Ct) values for determining the etiological relevance of detected pathogens.
Main Methods:
- Nucleic acid extraction and real-time PCR were performed on 466 samples from 632 Tanzanian children (<5 years) with and without gastrointestinal symptoms.
- Samples were analyzed from direct stool, Whatman papers, and flocked swabs stored at ambient temperature.
- Pathogen detection frequencies and cycle threshold (Ct) values were compared across different sample types.
Main Results:
- Real-time PCR achieved equal or higher frequencies of pathogen detection from Whatman papers and flocked swabs compared to direct stool extraction.
- No significant differences in Ct values were observed based on the nucleic acid extraction strategy.
- Ct values did not reliably indicate whether a detected pathogen was associated with gastrointestinal symptoms.
Conclusions:
- Whatman papers and flocked swabs are suitable and effective matrices for storing and transporting stool samples for real-time PCR-based pathogen detection in tropical settings.
- The cycle threshold (Ct) value alone is insufficient for determining the clinical significance or etiological relevance of detected pathogens in this context.
- These findings support the use of simplified sample preservation methods to enhance molecular diagnostic capabilities in resource-limited tropical areas.
Abstract:
The study was performed to compare real-time PCR after nucleic acid extraction directly from stool samples as well as from samples stored and transported on Whatman papers or flocked swabs at ambient temperature in the tropics. In addition, the possible suitability for a clear determination of likely aetiological relevance of PCR-based pathogen detections based on cycle threshold (Ct) values was assessed. From 632 Tanzanian children <5 years of age with and without gastrointestinal symptoms, 466 samples were subjected to nucleic acid extraction and real-time PCR for gastrointestinal viral, bacterial and protozoan pathogens. Equal or even higher frequencies of pathogen detections from Whatman papers or flocked swabs were achieved compared with nucleic acid extraction directly from stool samples. Comparison of the Ct values showed no significant difference according to the nucleic acid extraction strategy. Also, the Ct values did not allow a decision whether a detected pathogen was associated with gastrointestinal symptoms.
