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. 2021 May 12;11(5):1369.
doi: 10.3390/ani11051369.

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

Pamela Carolina Köster  1 , Begoña Bailo  1 , Alejandro Dashti  1 , Carolina Hernández-Castro  1   2 , Rafael Calero-Bernal  3 , Francisco Ponce-Gordo  4 , David González-Barrio  1 , David Carmena  1
Affiliations

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

Pamela Carolina Köster et al. Animals (Basel). .

Abstract

Preservation and conservation of biological specimens, including faecal samples, is a challenge in remote areas or poor-resource settings where the cold chain cannot be maintained. This study aims at evaluating the suitability of filter cards for long-term storage of faecal samples of animal and human origin positive to the diarrhoea-causing protozoan parasites, Giardia duodenalis and Cryptosporidium hominis. Three commercially available Whatman® Filter Cards were comparatively assessed: the FTA® Classic Card, the FTA® Elute Micro Card, and the 903 Protein Saver Card. Human faecal samples positive to G. duodenalis (n = 5) and C. hominis (n = 5) were used to impregnate the selected cards at given storage (1 month, 3 months, and 6 months) periods and temperature (-20 °C, 4 °C, and room temperature) conditions. Parasite DNA was detected by PCR-based methods. Sensitivity assays and quality control procedures to assess suitability for genotyping purposes were conducted. Overall, all three Whatman® cards were proven useful for the detection and molecular characterisation of G. duodenalis and C. hominis under the evaluated conditions. Whatman® cards represent a simple, safe, and cost-effective option for the transportation, preservation, and storage of faecal samples without the need of the cold chain.

Keywords: Cryptosporidium hominis; Giardia duodenalis; PCR; faeces; filter card; preservation; storage; transportation.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Standardisation of sampling areas for each Whatman® card used in the present study to assess storage periods and conditions of impregnated stool samples (a) FTA® Classic Card; (b) FTA® Elute Micro Card; (c) 903 Protein Saver Card.
Figure 2
Figure 2
Box plot of cycle threshold (Ct) values generated from Giardia duodenalis isolates extracted from the three Whatman® cards evaluated in the present study at different storage conditions. (a): 1 month-length storage; (b): 3 months-length storage; (c): 6 month-length storage. Horizontal thick lines within boxes represent median values Upper and lower whiskers represent the data range. Plotted dots represent outliers. Using the Tukey’s Honestly Significant Difference test as multiple post hoc comparison method, statistical significance is represented as * (p < 0.05).
Figure 3
Figure 3
Agarose gel electrophoresis (2% w/v) detection of PCR products showing the presence of a 587-bp amplicon for the Cryptosporidium hominis ssu rRNA gene extracted from the three Whatman® cards evaluated in the present study at different storage conditions. (a): 1 month-length storage; (b): 3 months-length storage; (c): 6 month-length storage.
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