Whatman Protein Saver Cards for Storage and Detection of Parasitic Enteropathogens

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 realtime 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.

Long-Term Preservation and Storage of Faecal Samples in Whatman® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis

“…This tool has been also assessed for the molecular detection of gastrointestinal parasites (e.g., the coccidian Cryptosporidium spp. and Cyclospora cayetanensis , the flagellated G. duodenalis , and the microsporidia Encephalitozoon intestinalis ) in matrices including clinical specimens and fresh produce [ 16 , 17 , 18 ]. In a seminal study, FTA ® card templates prepared from purified Cryptosporidium spp.…”

“…Additionally, Whatman ® cards have been proven useful for the diagnosis of pathogenic blood parasites, including canine microfilariae [ 14 ] and malaria-causing Plasmodium species [ 15 ]. However, very few studies have attempted to evaluate their efficacy for the detection of enteric pathogens in stool samples [ 16 , 17 , 18 ]. The protozoan enteroparasites, Giardia duodenalis (syn.…”

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

“…In a study in Bangladesh, similarly good results were shown for the preservation of DNA of protozoan pathogens on Whatman 903 protein saver cards (Sigma‐Aldrich, St. Louis, MO) in a real‐life diagnostic setting (Natarajan et al . 2018). A long‐term assessment confirmed stability of bacterial DNA on Whatman FTA papers stored at ambient temperature in a British laboratory for three years under experimental conditions (Rajendram et al .…”

Use of Chelex-100 for the molecular diagnosis of five animal pathogens

“…Although the first nucleic acid isolation was achieved 150 years ago, there is no general Gold-Standard for downstream applications. Chelex-100 resin has been used since the '90s for diagnostic (Noh et al 2019;, Chen et al 1991) and other purposes (Gill et al 1992) given that it is a rapid, easily performed, and non-expensive method from a variety of biological samples such as semen, blood, tissues, and milk among others, with different sensitivities depending on the pathogen (Natarajan et al 2018).…”