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Whatman Protein Saver Cards for Storage and Detection of Parasitic Enteropathogens
Gayathri Natarajan1, Mamun Kabir2, Jamie Perin1
1Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland.
Abstract:
Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards. DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction. For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.
Insights
A low-cost method using Whatman 903 protein saver cards effectively preserves stool for detecting Giardia and Entamoeba histolytica in low-resource settings. This method offers high sensitivity and specificity for key parasitic enteropathogens.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Global Health
Background:
- Diarrhea etiology identification is hindered by laboratory storage needs, especially in low-resource settings.
- Preserving stool specimens for parasitic enteropathogen detection remains a challenge.
Purpose of the Study:
- To evaluate the efficacy of Whatman 903 protein saver cards for preserving stool specimens.
- To assess the low-cost preservation method for detecting parasitic enteropathogens like Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica.
Main Methods:
- 232 stool samples positive for Giardia, Cryptosporidium, and Entamoeba histolytica were preserved on Whatman 903 cards.
- DNA extraction was performed using Chelex and Qiagen protocols, followed by multiplex real-time PCR.
- Sensitivity and specificity were determined by comparing results with direct DNA extraction from whole stool samples.
Main Results:
- For samples with high parasite load (ct < 30), Whatman card preservation showed 100% sensitivity for Giardia with both extraction methods.
- Entamoeba histolytica detection achieved 100% sensitivity with Qiagen extraction and 80% with Chelex.
- Cryptosporidium detection sensitivity was 80% for Qiagen and 50% for Chelex; specificity was 100% for all parasites.
Conclusions:
- Whatman 903 protein saver cards are effective for preserving stool specimens for Giardia and Entamoeba histolytica detection.
- The method is recommended for use with Qiagen DNA extractions in low-resource settings due to high sensitivity and specificity.
- This low-cost approach can improve parasitic enteropathogen diagnostics in resource-limited environments.
